Megakaryocytic protein tyrosine kinases

ABSTRACT

The present invention relates to novel cytoplasmic tyrosine kinases isolated from megakaryocytes (megakaryocyte kinases or MKKs) which are involved in cellular signal transduction pathways and to the use of these novel proteins in the diagnosis and treatment of disease. The present invention further relates to specific megakaryocyte kinases, designated MKK1, MKK2 and MKK3, and their use as diagnostic and therapeutic agents.

This application is a continuation-in-part application of U.S. application Ser. No. 08/232,545, filed Apr. 22, 1994, which is incorporated herein by reference.

TABLE OF CONTENTS

1. Introduction

2. Background

3. Summary of the Invention

4. Brief Description of the Figures

5. Detailed Description

5.1. The MKK Coding Sequences

5.2. Expression of MKK

5.3. Expression Systems

5.4. Identification of Transfectants or Transformants that Express the MKK

5.5. Uses of MKK and Engineered Cell Lines

5.5.1. Antibody Production and Screening

5.5.2. Screening of Peptide Library with MKK or MKK Engineered Cell Lines

5.5.3. Screening of Organic Compounds with MKK Protein or Engineered Cell Lines

5.6. Uses of MKK Polynucleotide

5.6.1. Diagnostic Uses of an MKK Polynucleotide

5.6.2. Therapeutic Uses of an MKK Polynucleotide

6. Examples: Cloning and Characterization of MKK1

6.1. cDNA Cloning, MKK Expression and MKK Characterization

6.1.1. Full-length cDNA Cloning

6.1.2. MKK Expression

6.1.3. RNA Blot Analysis of MKKs

7. Example: Autophosphorylation of MKK2 and MKK3

8. Example: Production of Anti-MKK Antibodies and Immunoprecipitation of MKK

9. Example: Expression of MKK1 Anti-sense Sequences

10. Example: MKKR Protein Tyrosine Kinase Activity

10.1. Materials and Methods .

10.2. Results

11. Example: Biological Activity of MKK1

11.1. Materials and Methods

11.1.1. Retroviral Infection of L-8057 cells

11.1.2. Cell Growth Measurement of MKK1 Expressing L-8057 Cells

11.1.3. Growth Factor Response of MKK1 Expressing L-8057 Cells

11.2. Results

11.2.1. Cell Growth

11.2.2. Cytokine Stimulation

11.2.3. Cell Differentiation

WHAT IS CLAIMED

ABSTRACT

1. Introduction

The present invention relates to novel cytoplasmic tyrosine kinases isolated from megakaryocytes (megakaryocyte kinases or MKKs) which are involved in cellular signal transduction pathways and to the use of these novel proteins in the diagnosis and treatment of disease.

The present invention further relates to specific megakaryocyte kinases, designated MKK1, MKK2 and MKK3, and their use as diagnostic and therapeutic agents.

2. Background

Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. These processes include, but are not limited to, cell proliferation, differentiation and survival. Many tyrosine kinases are expressed in postmitotic, fully differentiated cells, particularly in the case of hematopoietic cells, and it seems likely that these proteins are involved in specialized cellular functions that are specific for the cell types in which they are expressed. (Eiseman, E. and J. B. Bolen, Cancer Cells 2(10):303-310, 1990). A central feature of signal transduction is the reversible phosphorylation of certain proteins. (for reviews, see Posada, J. and Cooper, J. A., 1992, Mol. Biol. Cell 3:583-392; Hardie, D. G., 1990, Symp. Soc. Exp. Biol. 44:241-255). The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine kinases (TKs), which function to phosphorylate proteins, and tyrosine phosphatases (TPs), which function to dephosphorylate proteins. Normal cellular function requires a delicate balance between the activities of these two types of enzyme.

Phosphorylation of cell surface tyrosine kinases, stimulates a physical association of the activated receptor with intracellular target molecules. Some of the target molecules are in turn phosphorylated. Other target molecules are not phosphorylated, but assist in signal transmission by acting as adapter molecules for secondary signal transducer proteins.

The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular signal transduction include Aaronson, S. A., Science 254:1146-1153, 1991; Schlessinger, J. Trends Biochem. Sci. 13:443-447, 1988; and Ullrich, A., and Schlessinger, J. Cell 61:203-212, 1990.

Receptor tyrosine kinases are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. The intracellular, cytoplasmic, nonreceptor protein tyrosine kinases may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrane domain. Bolen (Oncogene, vol. 8, pgs. 2025-2031 (1993)) reports that 24 individual protein tyrosine kinases comprising eight different families of non-receptor protein tyrosine kinases have been identified: Abl/Arg; Jak1/Jak2/Tyk2; Fak; Fes/Fps; Syk/Zap; Tsk/Tec/Atk; Csk; and the Src group, which includes the family members Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. All of the non-receptor protein tyrosine kinases are thought to be involved in signaling pathways that modulate growth and differentiation. Bolen, supra, suggests that half of the nonreceptor protein tyrosine kinases have demonstrated oncogenic potential and half appear to be primarily related to suppressing the activity of Src-related protein kinases and could be classified as anti-oncogenes.

While distinct in their overall molecular structure, each member of a given morphotypic family of cytoplasmic protein tyrosine kinases shares sequence homology in certain non-catalytic domains in addition to sharing sequence homology in the catalytic kinase domain. Examples of defined non-catalytic domains include the SH2 (SRC homology domain 2; Sadowski, I et al., Mol. Cell. Biol. 6:4396-4408; Kock, C. A. et al., 1991, Science 252:668-674) domains, SH3 domains (Mayer, B. J. et al., 1988, Nature 332:269-272) and PH domains (Musacchio et al., TIBS 18:343-348 (1993). These non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson, T. and Gish, G., 1992, Cell 71:359-362).

While the metabolic roles of cytoplasmic protein tyrosine kinases are less well understood than that of the receptor-type protein tyrosine kinases, significant progress has been made in elucidating some of the processes in which this class of molecules is involved. For example, members of the src family, lck and fyn, have been shown to interact with CD4/CD8 and the T cell receptor complex, and are thus implicated in T cell activation, (Veillette, A. Davidson, D., 1992, TIG 8:61-66). Some cytoplasmic protein tyrosine kinases have been linked to certain phases of the cell cycle (Morgan, D. O. et al., 1989, Cell 57:775-786; Kipreos, E. T. et al., 1990, Science 248:217-220; Weaver et al., 1991, Mol. Cell. Biol. 11:4415-4422), and cytoplasmic protein tyrosine kinases have been implicated in neuronal and hematopoietic development (Maness, P., 1992, Dev. Neurosci 14:257-270 and Rawlings et al., Science 261:358-361 (1993)). Deregulation of kinase activity through mutation or overexpression is a well-established mechanism underlying cell transformation (Hunter et al., 1985, supra; Ullrich et al., supra).

A variety of cytoplasmic tyrosine kinases are expressed in, and may have important functions in, hematopoietic cells including src, lyn, fyn, blk, lck, csk and hck. (Eisenian, E. and J. B. Bolen, Cancer Cells 2(10):303-310, 1990). T-cell activation, for example, is associated with activation of lck. The signaling activity of lyn may be stimulated by binding of allergens to IgE on the surface of basophils. (Eisenian, supra).

Abnormalities in tyrosine kinase regulated signal transduction pathways can result in a number of disease states. For example, mutations in the cytoplasmic tyrosine kinase atk (also called btk) are responsible for the x-linked agammaglobulinemia, (Ventrie, D., et al., Nature 361:226-23, 1993). This defect appears to prevent the normal differentiation of pre-B cells to mature circulating B cells and results in a complete lack of serum immunoglobulins of all isotypes. The cytoplasmic tyrosine kinase Zap-70 has been suggested as indispensable for the development of CD8 single-positive T cells as well as for signal transduction and function of single-positive CD4 T cells, and lack of this protein leads to an immunodeficiency disease in humans, (Arpala, E., et al., Cell 76:1-20, 1994). Gene knockout experiments in mice suggest a role for src in the regulation of osteoclast function and bone remodeling as these mice develop osteopetrosis. (Soriano et al., Cell 64:693-702, 1991 and Lowe et al., PNAS (in press)).

Megakaryocytes are large cells normally present in bone marrow and spleen and are the progenitor cell for blood platelets. Megakaryocytes are associated with such disease states as acute megakaryocytic leukemia (Lu et al., Cancer Genet Cytogenet, 67(2):81-89 (1993) and Moody et al., Pediatr Radiol. 19(6-7):486-488 (1989)), a disease that is difficult to diagnose early and which is characterized by aberrant proliferation of immature cells or “blasts”; myelofibrosis (Smith et al., Crit Rev Oncol Hematol. 10(4):305-314 (1990) and Marino, J. Am. Osteopath Assoc. 10:1323-1326 (1989)), an often fatal disease where the malignant cell may be of megakaryocytic lineage and may be mediated by platelet or megakaryocyte growth factors; acute megakaryocytic myelosis (Fohlmeister et al., Haematologia 19(2):151-160 (1986)) a rapidly fatal disease characterized by megakaryocytic proliferation and the appearance of immature megakaryocytes in the circulation; and acute myelosclerosis (Butler et al., Cancer 49(12):2497-2499 (1982) and Bearman et al., Cancer 43(1):279-93 (1979)) a myeloproliferative syndrome where the marrow is characterized by atypical megakaryocytes.

Platelets play a key role in the regulation of blood clotting and wound healing, as well as being associated with such disease conditions as thrombocytopenia, atherosclerosis, restenosis and leukemia. Several receptor tyrosine kinases have been identified in human megakaryocytes including c-kit, blg and blk. (Hoffman, H., Blood 74:1196-1212, 1989; Long, M. W., Stem Cells 11:33-40, 1993; Zaebo, K. M., et al., Cell 63:213-224,1990). Cytoplasmic tyrosine kinases of human megakaryocytic origin have also been reported. (Bennett et al., Journal of Biological Chemistry 289(2):1068-1074, 1994; Lee et al., Gene 1-5, 1993; and Sakano et al., Oncogene 9:1155-1161 (1994)).

3. SUMMARY OF THE INVENTION

The present invention relates to novel, cytoplasmic tyrosine kinases isolated from megakaryocytes (megakaryocyte kinases or MKKs) which are involved in cellular signal transduction pathways. Particular MKKs described herein are referred to as MKK1, MKK2, and MKK3. The complete nucleotide sequences encoding MKK1, MKK2, and MKK3 are disclosed herein, and provide the basis for several aspects of the invention hereinafter described.

The present invention is based, in part, upon the discovery that MKK1, MKK2, and MKK3 have amino acid and structural homology, respectively, to the PTKs csk (Brauninger et al. Gene, 110:205-211 (1992) and Brauninger et al., Oncogene, 8:1365-1369 (1993)), atk/btk, tec and tsk (Vetrie et al., Nature 361:226-233 (1993); Mano et al., Oncogene 8:417-424 (1993) and Heyeck et al., PNAS USA 90:669-673,1993, respectively) and fyn (Kawakami et al. Mol. Cell. Bio. 6:4195-4201, 1986)).

The present invention also relates, in part, to nucleotide sequences and expression vectors encoding MKKs. Also described herein are methods of treatment and diagnosis of diseases resulting from abnormalities in signal transduction pathways in which MRKs are involved.

The MKK sequences disclosed herein may be used to detect and quantify levels of MKK mRNA in cells and furthermore for diagnostic purposes for detection of expression of MKKs in cells. For example, an MKK sequence may be used in hybridization assays of biopsied tissue to diagnose abnormalities in gene expression associated with a transformed phenotype.

Also disclosed herein are methods of treatment of diseases or conditions associated with abnormalities in signal transduction pathways in megakaryocytes. Such abnormalities can result in, for example, under production of mature, differentiated cells, inappropriate proliferation of immature cells or modulation of activity of other important cellular functions.

Anti-MKK antibodies may be used for diagnostic purposes for the detection of MKKs in tissues and cells. Anti-MKK antibodies may also be used for therapeutic purposes, for example, in neutralizing the activity of an MKK associated with a signal transduction pathway.

Oligonucleotide sequences, including anti-sense RNA and DNA molecules and ribozymes, designed to inhibit the translation of MKK mRNA, may be used therapeutically in the treatment of disease states associated with aberrant expression of MKKs. In a particular embodiment of the invention described by way of Example 9 herein, an anti-MKK1 antisense molecule is used to inhibit MKK-1 protein synthesis resulting in reduced megakaryocyte growth and differentiation.

Proteins, peptides and organic molecules capable of modulating activity of MKKs may be used therapeutically in the treatment of disease states associated with aberrant expression of MKKs. Alternatively, proteins, peptides and organic molecules capable of modulating activity of MKKs may be used therapeutically to enhance normal activity levels of MKKs. For example, small molecules found to stimulate MKK1 activity in megakaryocytes may be used for ex vivo culturing of megakaryocytes intended for autologous treatment of patients receiving chemotherapy or other therapies which deplete megakaryoctyes or platelets, or in the treatment of thrombocytopenia.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B. Human MKK1 nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2). Marked regions show the SH2 and SH3 domains, and the catalytic domain.

FIGS. 2A and 2B. Human MKK2 nucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4). Marked regions show the pleckstrin homology domain (PH), the proline rich sequences following the PH domain, the SH2 and SH3 domains, and the catalytic domain.

FIGS. 3A and 3B. Human MKK3 nucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO:6). Marked regions show the SH2 and SH3 domains, and the catalytic domain.

FIG. 4. Expression of MKK1 and MKK2 in human and rodent cell lines.

FIG. 5. Immunoprecipitation (i.p.) of in vitro transcribed and translated MKK1 and MKK2 proteins. Samples in lanes designated 1 through 9 are as follows: 1. MKK1 i.p. with anti-carboxy terminus MKK1 Ab, 2. and 3. MKK1 i.p. with anti-amino terminus MKK1 Ab, 4. MKK1 i.p. with rabbit pre immune sera, 5. MKK2 i.p. with rabbit pre immune sera, 6. and 7. MKK2 i.p. with anti-carboxy terminus MKK2 Ab, 8. MKK1 in vitro transcribed/translated protein without i.p., 9. MKK2 in vitro transcribed/translated protein without i.p.

FIGS. 6A and 6B. FIGS. 6A-6B illustrate anti-sense MKK1 expression suppresses AChE Production in primary murine bone marrow cultures. FIG. 6A illustrates AChE production. FIG. 6B illustrates MKK1 protein expression.

FIG. 7. MKK2 and MKK3 autophosphorylate and transphosphorylate proteins when expressed in bacteria. Lanes 2, 4, and 6 represent non-induced bacteria expressing MKK1, MKK2, MKK3, respectively. Lanes 1, 3, and 5 represent induced bacteria expressing MKK1, MKK2, MKK3, respectively.

FIG. 8. MKK expression constructs.

FIG. 9. Shared amino acid sequence homology of MKK1 (SEQ ID NO:2) and csk (SEQ ID NO:7).

FIGS. 10A and 10B. Shared amino acid sequence homology of MKK2 (SEQ ID NO:4) and atk/btk (hAtk is SEQ ID NO:8; hTKT is SEQ ID NO:9; and mTec is SEQ ID NO:10).

FIGS. 11A, 11B, 11C and 11D. Shared amino acid sequence homology of MKK3 (SEQ ID NO:6) and src tyrosine kinase family members (hAtk is SEQ ID NO:11; cYrk is SEQ ID NO:12; hSrc is SEQ ID NO:13; hYes is SEQ ID NO:14; hFgr is SEQ ID NO:15, hLyn is SEQ ID NO:16; hHck is SEQ ID NO:17; hLck is SEQ ID NO:18; and mBlk is SEQ ID NO:19).

FIG. 12. FIG. 12 illustrates that the hyperexpression of MKK-1 in L-8057 cells grown in serum-free media inhibits cell growth of those cells as compared to control L-8057 cells.

FIG. 13. FIG. 13 illustrates the stimulation of MKK-1 infected L-8057 cells and control L-8057 cells with rat stem cell factor and IL-3.

FIG. 14. FIG. 14 illustrates the effect of tetradecanoyl phorbol acetate (“TPA”) on either control cells or cells that express MKK-1.

5. DETAILED DESCRIPTION

The present invention relates to novel, cytosolic megakaryocytic kinases referred to herein as “MKKs”, and in particular to megakaryocyte kinase 1 (MKK1), megakaryocyte kinase 2 (MKK2), which are expressed in human megakaryocytic cell lines, and megakaryocyte kinase 3 (MKK3).

As used herein, MKK is a term which refers to MKK1, MKK2 and MKK3 from any species, including, bovine, ovine, porcine, equine, murine and preferably human, in naturally occurring-sequence or in variant form, or from any source, whether natural, synthetic, or recombinant. A preferred MKK variant is one having at least 80% amino acid homology, a particularly preferred MKK variant is one having at least 90% sequence homology and another particularly preferred MKK variant is one having at least 95% amino acid homology to the naturally occurring MKK.

MKK1 is a cytosolic tyrosine kinase of molecular weight 58 kD, as determined by SDS gel electrophoresis, having homology to the TK csk (Partanen, et al., Oncogene 6:2013-2018 (1991) and Nada et al., Nature 351:69-72 (1991)) in the intervening sequences of its catalytic domain, the SH2 and SH3 domains, and other non-catalytic regions and like csk, lacks regulatory phosphorylation sites corresponding to c-src tyrosines 416 and 527. MKK1 also lacks an amino-terminal myristylation site.

Csk is a recently described novel cytoplasmic TK that seems to play a key role in regulation of signal transduction in hematopoietic and neural development. For example csk has been shown to negatively regulate members of the src family of TKs, including c-src, lck, and fyn, through its ability to phosphorylate regulatory tyrosines. (Bergman et al., The EMBO Journal 11(8)8:2919-2924 (1992) and Sabe et al., Molecular and Cellular Biology 12(10):4706-4713 (1992)). Autero et al., (Molecular and Cellular Biology 14(2):1308-1321 (1994)) have reported that csk positively regulates a phosphatase, CD45, that is key to T-cell activity. Csk mediated phosphorylation of CD45 phosphotyrosine phosphatase (PTPase) caused a several fold increase in its PTPase activity. Csk appears to play a role as a regulator of the sequence of both phosphorylation and dephosphorylation events culminating in cell activation and proliferation.

Defective expression of csk in mouse embryos results in defects in the neural tube with subsequent death between day 9 and day 10 of gestation, with cells derived from these embryos exhibiting an order of magnitude increase in activity of src kinase (Nada et al., Cell 73:1125-1135 (1993)). Overexpression of csk in transformed rat 3Y1 fibroblasts was shown to cause reversion to normal phenotypes (Sabe et al., Molecular and Cellular Biology 12:4706-4713 (1992)).

MKK1 has 54% homology with csk at the amino acid level and structural similarity to csk, i.e., the lack of regulatory phosphorylation sites and the lack of an amino-terminal myristylation site. Experimental data, see Section 9, show that expression of human anti-sense MKK1 sequences inhibits synthesis of murine MKK1, which inhibition is associated with a reduction of proliferation of megakaryocytes in vitro. Based upon the experimental data in Section 9 and amino acid and structural homology with csk, MKK1 appears to play a regulatory role in the growth and differentiation of megakaryocytes and perhaps neural tissues based on its expression in those tissues.

MKK2 is a novel cytosolic tyrosine kinase of molecular weight 78 kD, as determined by SDS gel electrophoresis, having homology to the tec subfamily of TKs which also includes tsk and atk/btk. Like the tec subfamily, MKK2 lacks an amino-terminal site for myristylation and has a putative pleckstrin homology binding domain located 5′ to the SH3 domain (Musacchio et al., TIBS 18:343-348 (1993)). The pleckstrin homology (PH) domain has been found in a number of proteins with diverse cellular functions and is abundant in proteins involved in signal transduction pathways. Musacchio et al., supra suggest that the PH domain may be involved in molecular recognition similarly to SH2 and SH3 domains.

The tec family of tyrosine kinases appear to play roles in cellular differentiation and include family members tec, a kinase which may be specifically involved in the cell growth of hepatocytes or hepatocarcinogenesis (Mano et al., supra); tsk, which may play a role in early T-lymphocyte differentiation (Heyek et al., PNAS USA 90:669-673 (1993)) and atk/btk. Aberrant expression of atk/btk has been shown to be responsible for X-linked agammaglobulinemia (XLA), a human disease resulting from a developmental block in the transition from pre-B cells to mature B cells (Ventrie, D. et al., supra).

MKK2 has 50% homology to atk/btk at the amino acid level and structural similarity to tec family members, i.e., the presence of the SH2, SH3 and PH domains and the lack of an amino-terminal site for myristylation and the carboxyl site of tyrosine phosphorylation found in family members. Based upon the amino acid homology and structural similarity to tec family members which play roles in cellular differentiation, MKK2 may play a role in the differentiation of megakaryoctyes.

NKK3 is a novel cytosolic tyrosine kinase of molecular weight 58 kD, as determined by SDS gel electrophoresis, having homology to the TK fyn. MKK3 does not have a myristylation sites. MKK3 does have a putative regulatory cite at tyr 387 but the surrounding 12 amino acids are not identical with other members of the src subfamily that share highly conserved sequences in this region. MKK3 has 47% homology with fyn at the amino acid level.

The fyn gene was originally characterized in normal human fibroblast and endothelial cells, but it is also expressed in a variety of other cell types. Alternative splicing of fyn has been shown to yield two distinct transcripts, both coding for enzymatically active forms of the kinases.

MKK sequences could be used diagnostically to measure expression of MKKs in disease states, such as for example leukemia, where abnormal proliferation of immature myeloid cells occurs, or where abnormal differentiation of megakaryocytes occurs. MKKs could also be used therapeutically in the treatment of disease states involving abnormal proliferation or differentiation through interruption of signal transduction by modulation of protein tyrosine kinases.

The nucleotide and deduced amino acid sequence of human MKK1, MKK2, and MKK3 are shown in FIGS. 1A-1B, 2A-2B and 3A-3B, respectively. FIGS. 9, 10A-10B and 11A-11D show the shared sequence homology between MKKs and related tyrosine kinases.

5.1. The MKK Coding Sequences

The nucleotide coding sequence and deduced amino acid sequence of the human MKK1, MKK2, and MKK3 genes are depicted in FIGS. 1A-1B, 2A-2B and 3A-3B, respectively. In accordance with the invention, any nucleotide sequence which encodes the amino acid sequence of an MKK gene product can be used to generate recombinant molecules which direct the expression of an MKK.

In a specific embodiment described herein, the human MKK1, MKK2, and MKK3 genes were isolated by performing polymerase chain reactions (PCR) in combination with two degenerate oligonucleotide primer pools that were designed on the basis of highly conserved sequences within the kinase domain of receptor tyrosine kinases corresponding to the amino acid sequence HRDLAA (sense primer) and SDVWS/FY (antisense primer) (Hanks et al., 1988). The MKK cDNAs were synthesized by reverse transcription of poly-A RNA from the human K-562 cell line, ATCC accession number CCL 243, or from the Meg 01 cell line, (Ogura et al., Blood 66:1384 (1985)).

The PCR fragments were used to screen a lambda gt11 library of human fetal brain. For each individual MKK, several overlapping clones were identified. The composite of the cDNA clones for MKK1, MKK2, and MKK3 are depicted in FIGS. 1A-1B, 2A-2B, and 3A-3B, respectively.

Further characterization of the individual MKKs is found infra.

5.2. Expression of MKK

In accordance with the invention, MKK polynucleotide sequences which encode MKKs, peptide fragments of MKKs, MKK fusion proteins or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of MKK protein, MKK peptide fragment, fusion proteins or a functional equivalent thereof, in appropriate host cells. Such MKK polynucleotide sequences, as well as other polynucleotides which selectively hybridize to at least a part of such MKK polynucleotides or their complements, may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc.

Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of the MKK protein. Such DNA sequences include those which are capable of hybridizing to the human MKK sequence under stringent conditions. The phrase “stringent conditions” as used herein refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium citrate at 42° C; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M Sodium citrate), 5×Denhardt's solution, sonicated salmon sperm DNA 150 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within an MKK sequence, which result in a silent change thus producing a functionally equivalent NKK. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipatic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

The DNA sequences of the invention may be engineered in order to alter an MKK coding sequence for a variety of ends including but not limited to alterations which modify processing and expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.

In another embodiment of the invention, an MKK or a modified MKK sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for inhibitors of MKK activity, it may be useful to encode a chimeric MKK protein expressing a heterologous epitope that is recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between an MKK sequence and the heterologous protein sequence, so that the MKK may be cleaved away from the heterologous moiety.

In an alternate embodiment of the invention, the coding sequence of an MKK could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817. Alternatively, the protein itself could be produced using chemical methods to synthesize an MKK amino acid sequence in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. (e.g., see Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins. Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49.

In order to express a biologically active MKK, the nucleotide sequence coding for MKK, or a functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. The MKK gene products as well as host cells or cell lines transfected or transformed with recombinant MKK expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies (i.e., monoclonal or polyclonal) that competitively inhibit activity of an MKK and neutralize its activity. Anti-MKK antibodies may be used in detecting and quantifying expression of an MKK in cells and tissues.

5.3. Expression Systems

Methods which are well known to those skilled in the art can be used to construct expression vectors containing an MKK coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. see, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to express an MKK coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an MKK coding sequence; yeast transformed with recombinant yeast expression vectors containing an MKK coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an MKK coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an MKK coding sequence; or animal cell systems. The expression elements of these systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell lines that contain multiple copies of an MKK DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.

In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the MKK expressed. For example, when large quantities of MKK1 are to be produced for the generation of antibodies, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the MKK1 coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid AS-lac Z protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.

In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, 1987, Acad. Press, N.Y. 153:516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y. 152:673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II.

In cases where plant expression vectors are used, the expression of an MKK coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984, Science 224:838-843); or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

An alternative expression system which could be used to express an MKK is an insect system. In one such system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. An MKK coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter). Successful insertion of an MKK coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (e.g., see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, an MKK coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing an MKK in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may be used. (See, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

Specific initiation signals may also be required for efficient translation of an inserted MKK coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire MKK gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of an MKK coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of an MKK coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express an MKK may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with MKK DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express an MKK.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981), Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Ed.).

5.4. Identification of Transfectants or Transformants that Express the MKK

The host cells which contain the coding sequence and which express the biologically active gene product may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of “marker” gene functions; (c) assessing the level of transcription as measured by the expression of MKK mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity.

In the first approach, the presence of the MKK coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the MKK coding sequence, respectively, or portions or derivatives thereof.

In the second approach, the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain “marker” gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the MKK1 coding sequence is inserted within a marker gene sequence of the vector, recombinant cells containing the MKK1 coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with an MKK sequence under the control of the same or different promoter used to control the expression of the MKK coding sequence. Expression of the marker in response to induction or selection indicates expression of the MKK coding sequence.

In the third approach, transcriptional activity for an MKK coding region can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to an MKK coding sequence or particular portions thereof. Alternatively, total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of an MKK protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.

5.5. Uses of MKK and Engineered Cell Lines

Megakaryocytes, the progenitor cell for blood platelets, and platelets are associated with disease states involving aberrant proliferation or differentiation of such cells, such as acute megakaryocytic leukemia, acute megakaryocytic myelosis and thrombocytopenia. MKKs appear to play a role in the growth and differentiation of megkaryocytes, therefore inhibitors of MKKs may be used therapeutically for the treatment of disease states resulting from aberrant growth of megakaryocytes or platelets. Alternatively, enhancers of MKKs may be used therapeutically to stimulate the proliferation of megakaryocytes in such applications as, for example, ex vivo culturing of megakaryocytes intended for autologous cell therapy in individuals receiving chemotherapy or other therapies which deplete megakaryocytes or platelets or in treating thrombocytopenia caused by other conditions.

In an embodiment of the invention, an MKK and/or cell line that expresses an MKK may be used to screen for antibodies, peptides, or other molecules that act as agonists or antagonists of MKK through modulation of signal transduction pathways. For example, anti-MKK antibodies capable of neutralizing the activity of MKK may be used to inhibit an MKK associated signal transduction pathway. Such antibodies can act intracellularly utilizing the techniques described in Marasco et al.(PNAS 90:7889-7893 (1993)) for example or through delivery by liposomes. Alternatively, screening of organic or peptide libraries with recombinantly expressed MKK protein or cell lines expressing MKK protein may be useful for identification of therapeutic molecules that function by modulating the kinase activity of MKK or its associated signal transduction pathway. A therapeutic molecule may find application in a disease state associated with megakaryocytes, such as acute megakaryocytic leukemia, or alternatively, in non-disease applications, for example in ex vivo culturing of megakaryocytes intended for autologous treatment of individuals undergoing chemotherapy. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways deemed to be routine to those of skill in the art.

The ability of antibodies, peptides, or other molecules to prevent or mimic, the effect of MKK on signal transduction responses on MKK expressing cells may be measured. For example, responses such as activation or inhibition of MKK kinase activity or modulation of second messenger production may be monitored. The term “second messenger” as used herein refers to any component or product found in the cascade of signal transduction events. These assays may be performed using conventional techniques developed for these purposes.

5.5.1. Antibody Production and screening

Various procedures known in the art may be used for the production of antibodies to epitopes of the recombinantly produced MKK. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies, i.e., those which inhibit the biological activity, i.e., the kinase activity, of an MKK are especially preferred for diagnostics and therapeutics.

For the production of antibodies, various host animals may be immunized by injection with an MKK protein including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to an MKK may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce an MKK-specific single chain antibodies.

Antibody fragments which contain specific binding sites of an MKK may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the MKK of interest.

5.5.2. Screening of Peptide Library with MKK or HKK Engineered Cell Lines

Random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to MKK binding sites, e.g., SH2, SH3 or PH binding sites, or other functional domains of an MKK, such as kinase domains. The screening of peptide libraries may have therapeutic value in the discovery of pharmaceutical agents that act to stimulate or inhibit the biological activity of an MKK.

Identification of molecules that are able to bind to an MKK may be accomplished by screening a peptide library with recombinant MKK protein. Methods for expression of an MKK are described in Section 5.2, 5.3 and 5.4 and may be used to express a recombinant full length MKK or fragments of an MKK depending on the functional domains of interest. For example, the kinase and SH2, SH3 or PH binding domains of an MKK may be separately expressed and used to screen peptide libraries.

To identify and isolate the peptide/solid phase support that interacts and forms a complex with an MKK, it is necessary to label or “tag” the MKK molecule. The MKK protein may be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase or to other reagents such as fluorescent labels which may include fluorescein isothyiocynate (FITC), phycoerythrin (PE) or rhodamine. conjugation of any given label to MKK may be performed using techniques that are routine in the art. Alternatively, MKK expression vectors may be engineered to express a chimeric MKK protein containing an epitope for which a commercially available antibody exists. The epitope specific antibody may be tagged using methods well known in the art including labeling with enzymes, fluorescent dyes or colored or magnetic beads.

The “tagged” MKK conjugate is incubated with the random peptide library for 30 minutes to one hour at 22° C. to allow complex formation between an MKK and peptide species within the library. The library is then washed to remove any unbound MKK protein. If MKK has been conjugated to alkaline phosphatase or horseradish peroxidase the whole library is poured into a petri dish containing a substrate for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3′,4,4″-diamnobenzidine (DAB), respectively. After incubating for several minutes, the peptide/solid phase-MKK complex changes color, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescent tagged MKK molecule has been used, complexes may be isolated by fluorescent activated sorting. If a chimeric MKK protein expressing a heterologous epitope has been used, detection of the peptide/MKK complex may be accomplished by using a labeled epitope specific antibody. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing.

5.5.3. Screening of Organic Compounds with XKK Protein or Engineered Cell Lines

Cell lines that express an MKK may be used to screen for molecules that modulate MKK activity or signal transduction. Such molecules may include small organic or inorganic compounds or extracts of biological materials such as plants, fungi, etc., or other molecules that modulate MKK activity or that promote or prevent MKK mediated signal transduction. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways.

The ability of a test molecule to interfere with MKK signal transduction may be measured using standard biochemical techniques. Other responses such as activation or suppression of catalytic activity, phosphorylation or dephosphorylation of other proteins, activation or modulation of second messenger production, changes in cellular ion levels, association, dissociation or translocation of signalling molecules, or transcription or translation of specific genes may also be monitored. These assays may be performed using conventional techniques developed for these purposes in the course of screening. (See, for example, Peralidi, et al., J. Biochem. 285:71-78 (1992) or Campbell et al., JBC 268:7427-7434 (1993)).

Cellular processes under the control of an MKK signalling pathway may include, but are not limited to, normal cellular functions such as proliferation or differentiation of megakaryocytes or platelets, in addition to abnormal or potentially deleterious processes such as unregulated or inappropriate cell proliferation, blocking of differentiation of megakaryocytes or platelets, or ultimately cell death. The qualitative or quantitative observation and measurement of any of the described cellular processes by techniques known in the art may be advantageously used as a means of scoring for signal transduction in the course of screening.

MKK, or functional derivatives thereof, useful in identifying compounds capable of modulating signal transduction may have, for example, amino acid deletions and/or insertions and/or substitutions as long as they retain significant ability to interact with some or all relevant components of a MKK signal transduction pathway. A functional derivative of MKK may be prepared from a naturally occurring or recombinantly expressed MKK by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art. Alternatively, the functional derivative may be produced by recombinant DNA technology by expressing parts of MKK which include the functional domain in suitable cells. Functional derivatives may also be chemically synthesized. Cells expressing MKK may be used as a source of MKK, crude or purified for testing in these assays.

MKK signal transduction activity may be measured by standard biochemical techniques or by monitoring the cellular processes controlled by the signal. To assess modulation of kinase activity, the test molecule is added to a reaction mixture containing MKK and a substrate. The kinase reaction is then initiated with the addition of ATP. An immunoassay using an antiphosphotyrosine antibody is performed on the kinase reaction to detect the presence or absence of the phosphorylated tyrosine residues on the substrate or to detect phosphorylated tyrosine residues on autophosphorylated MKK, and results are compared to those obtained for controls i.e., reaction mixtures not exposed to the test molecule.

5.6. Uses of MKK Polynucleotide

An MKK polynucleotide may be used for diagnostic and/or therapeutic purposes. For diagnostic purposes, an MKK polynucleotide may be used to detect MKK gene expression or aberrant MKK gene expression in disease states, e.g., acute megakaryocytic leukemia or acute megakaryocytic myelosis. Included in the scope of the invention are oligonucleotide sequences, that include antisense RNA and DNA molecules and ribozymes, that function to inhibit translation of an MKK. In a specific embodiment of this aspect of the invention, an anti-MKK1 antisense molecule is shown to inhibit MKK-1 protein synthesis resulting in reduced megakaryocyte growth and differentiation.

5.6.1. Diagnostic Uses of an MKK Polynucleotide

An MKK polynucleotide may have a number of uses for the diagnosis of diseases resulting from aberrant expression of MKK. For example, the MKK1 DNA sequence may be used in hybridization assays of biopsies or autopsies to diagnose abnormalities of MKK1 expression; e.g., Southern or Northern analysis, including in situ hybridization assays. Such techniques are well known in the art, and are in fact the basis of many commercially available diagnostic kits.

5.6.2. Therapeutic Uses of an MKK Polynucleotide

An MKK polynucleotide may be useful in the treatment of various abnormal conditions. By introducing gene sequences into cells, gene therapy can be used to treat conditions in which the cells do not proliferate or differentiate normally due to underexpression of normal MKK or expression of abnormal/inactive MKK. In some instances, the polynucleotide encoding an MKK is intended to replace or act in the place of a functionally deficient endogenous gene. Alternatively, abnormal conditions characterized by overproliferation can be treated using the gene therapy techniques described below.

Abnormal proliferation of megakaryocytes is an important component of a variety of disease states such as acute megakaryocytic leukemia, myelofibrosis, or acute megakaryocytic myelosis. Recombinant gene therapy vectors, such as viral vectors, may be engineered to express variant, signalling incompetent forms of MKK which may be used to inhibit the activity of the naturally occurring endogenous MKK. A signalling incompetent form may be, for example, a truncated form of the protein that is lacking all or part of its catalytic domain. Such a truncated form may participate in normal binding to a substrate but lack enzymatic activity. Thus recombinant gene therapy vectors may be used therapeutically for treatment of diseases resulting from aberrant expression or activity of an MKK. Accordingly, the invention provides a method of inhibiting the effects of signal transduction by an endogenous MKK protein in a cell comprising delivering a DNA molecule encoding a signalling incompetent form of the MKK protein to the cell so that the signalling incompetent MKK protein is produced in the cell and competes with the endogenous MKK protein for access to molecules in the MKK protein signalling pathway which activate or are activated by the endogenous MKK protein.

Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of recombinant MKK into the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing an MKK polynucleotide sequence. See, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. Alternatively, recombinant MKK molecules can be reconstituted into liposomes for delivery to target cells.

Oligonucleotide sequences, that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of an MKK mRNA are within the scope of the invention. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between −10 and +10 regions of an MKK nucleotide sequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of MKK1 RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5′ and/or 3′ ends of the molecule or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

Methods for introducing polynucleotides into such cells or tissue include methods for in vitro introduction of polynucleotides such as the insertion of naked polynucleotide, i.e., by injection into tissue, the introduction of an MKK polynucleotide in a cell ex vivo, i.e., for use in autologous cell therapy, the use of a vector such as a virus, retrovirus, phage or plasmid, etc. or techniques such as electroporation which may be used in vivo or ex vivo.

6. EXAMPLES

Cloning and Characterization of MKK1

For clarity of discussion, the subsection below describes the isolation and characterization of a cDNA clone encoding the novel tyrosine kinase designated MKK1. The MKK2 and MKK3 genes were cloned and characterized using the same methods.

6.1. cDNA Cloning, MKK Expression and MKK Characterization

Confluent plates of K-562 cells (ATCC accession number CCL 243) were lysed by treatment with guanidinium-thiocyanate according to Chirgwin et al. (1979, Biochemistry 18:5294-5299). Total RNA was isolated by CsCl-gradient centrifugation. First-strand cDNA was synthesized from 20 μg total RNA with avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim).

cDNA was used in a polymerase chain reaction under standard conditions (PCR Technology-Principles and Applications for DNA Amplifications, H. E. Erlich, Ed., Stockton Press, New York 1989). Degenerate pools of primers corresponding to the amino acid sequence HRDLAA and SDVWSF/Y were prepared and used for the amplification:

5′ oligo pool

(SEQ ID NO:20)              H   R   D   L   A   A 5′ GGAATTCC CAC AGN GAC TTN GCN GCN AG 3′               T C   A T C   A   A   C

3′ oligo pool

(SEQ ID NO:21)             F/Y  S   W   V   D    S 5′ GGAATTCC GAA NGT CCA NAC GTC NGA 3′             ATG  CA          C    C

Thirty-five PCR cycles were carried out using 8 μg (0.8 μg) of the pooled primers. (Annealing 55° C., 1 min; Extension 72° C., 2 min; Denaturation 94° C., 1 min). The reaction product was subjected to polyacrylamide gel electrophoresis. Fragments of the expected size (˜210 bp) were isolated, digested with the restriction enzyme EcoRI, and subcloned into the pBluskript vector (Stratagene) using standard techniques (Current Protocols in Molecular Biology, eds. F. M. Ausubel et al., John Wiley & Sons, New York, 1988).

The recombinant plasmids were transformed into the competent E. coli strain designated 298.

The subcloned PCR products were sequenced by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463-5467) using Sequenase (United States Biochemical, Cleveland, Ohio 44111 USA). Clones designated MKK1, MKK2, and MKK3 were identified as novel TKs.

6.1.1. Full-length cDNA Cloning

The partial cDNA sequence of the new MKK1 TK, which was identified by PCR, was used to screen a λgt11 library from human fetal brain cDNA (Clontech) (complexity of 1×10¹⁰ recombinant phages). One million independent phage clones were plated and transferred to nitrocellulose filters following standard procedures (Sambrook, H. J., Molecular Cloning, Cold Spring Harbor Laboratory Press, USA, 1989). The filters were hybridized to the EcoRI/EcoRI fragment of clone MKK1, which had been radioactively labeled using 50 μCi [α³²P]ATP and the random-primed DNA labeling kit (Boehringer Mannheim). The longest cDNA insert of ˜3500 bp was digested with the restriction enzymes EcoRI/SacI to obtain a 5′ end probe of 250 bp. This probe was used to rescreen the human fetal brain library and several overlapping clones were isolated. The composite of the cDNA clones of MKK1, MKK2 and MKK3 is shown in FIGS. 1A-1B, 2A-2B and 3A-3B, respectively. The 1.75 million independent phage clones of a human placenta library, λZAP, were plated and screened with the 5′ end probe (EcoRI/SacI) of the clone used above. Subcloning of positive bacteriophages clones into pBluskript vector was done by the in vivo excision protocol (Stratagene).

The composite cDNA sequence and the predicted amino acid sequence of MKK1, MKK2 and MKK3 are shown in FIGS. 1A-1B, 2A-2B and 3A-3B, respectively.

6.1.2. MKK Expression

E. coli expression constructs for MKK1, MKK2 and MKK3 were produced by cloning of the corresponding cDNA fragments into a plasmid expression vector pTZS2 (Ray et al., PNAS USA 89:(13):5705-5709 (1992)) by substitution of recoverin coding sequence with synthetic polylinker fragment. To provide in-frame connection of the coding sequences to prokaryotic translation initiation site coded by the vector, an NdeI restriction site overlapping start codon (CATATG) was introduced in all three MKK cDNAs by site directed mutagenesis. The resulting constructs are designed to drive expression of unfused proteins with authentic amino acid sequences. FIG. 8 shows MRK expression constructs.

6.1.3. RNA Blot Analysis of MKXs

Total RNA was isolated from human megakaryocytes, myeloid cells, B-cells, T-cells, and epithelial cells.

PolyA⁺ RNA was isolated on an oligo (dT) column (Aviv and Leder, 1972, Proc. Natl. Acad. Sci. USA 69, 1408-1412). The poly A+ RNA was isolated using RNA stat −60 method (Tel-Test B Inc.) and blotted on a nitrocellulose filter using a slot blot apparatus (Schleicher and Schuell). 2 μg of poly A⁺ RNA was loaded per lane. The filter was hybridized with a ³²P-labeled EcoRI/EcoRI DNA fragment obtained by PCR. Subsequently, the filter was exposed to x-ray film at −70° C. with an intensifying screen. The results, as shown in FIG. 4, suggest that MKK1 and MKK2 are preferentially expressed in megakaryocytes. MKK3 expression could not be detected using this technique. FIG. 8 shows MKK expression constructs.

7. EXAMPLE

Autophosphorylation of MKK2 and KKK3

FIG. 7 represents Western blot analysis of protein from bacteria expressing MKK1, MKK2, or MKK3 using an anti-phosphotyrosine antibody (Hansen et al., Electrophoresis 14:112-126 (1993)). All MKK constructs were cloned into the inducible vector pTZS2, and transformed bacteria were grown under induced and uninduced conditions as described by Ray, et al.,(PNAS USA 89:5705-5709 (1992)). Bacterial pellets from these cultures were resuspended in sample buffer, containing 2-mercaptoethanol and SDS, and boiled. Proteins were separated by SDS-polyacrylamide gel electrophoresis. The results of this example indicate that MKK2 and MKK3 have kinase activity.

8. EXAMPLE

Production of Anti-MKK Antibodies and Immunoprecipitation of MKK

Antibodies recognizing MKK1 and MKK2 protein were made in rabbits using standard procedures. The anti-carboxy terminus MKK1 antibody was generated using the synthetic peptide GQDADGSTSPRSQEP which corresponds to residues 493-507 of amino acid sequence SEQ ID NO:2. The amino-terminus MKK1 Ab was generated using a GST-fusion proteins containing 78 amino acids coded by the SmaI to BG12 fragment of the MKK1 gene. The anti-carboxy terminus MKK2 Ab was made using a synthetic peptide corresponding to the sequence QQLLSSIIEPLREKDKH which is residues 660-675 of amino acid sequence SEQ ID NO:4.

MKK1 and MKK2, cloned into the pBluskript plasmid, were transcribed and translated in the presence of ³⁵S-methione using standards methods. Following protein synthesis MKK1 and MKK2 were immunoprecipitated (i.p.) with the appropriate rabbit antibodies (Ab) in the presence of SDS. FIG. 5 shows immunoprecipitation of in vitro transcribed and translated MKK1 and MKK2 proteins.

9. EXAMPLE

Expression of MKK1 Anti-sense sequences

Bone Marrow elements isolated from mice treated with 5-flurourocil 6 days prior to harvest were infected with retroviruses containing constructs expressing MKK1, antisense MKK1 (a truncated 5′ EcoR1-PvuII fragment cloned in the reverse orientation) or the empty retroviral vector (mock). Following infection, cells were cultured and analyzed for the level of acetylcholinesterase (AChE) as previously described, measured as optical density at 414 nm (Hill, Exp. Hematology 20:354-360 (1992). A higher optical density reading indicates a greater AChE level and correlates with increased megakaryocyte growth and differentiation. Levels of the murine MKK1 protein were determined by metabolically labeling cells with ³⁵S-methionine for 12 hours at the end of the experimental period. Following labeling, cells were lysed and MKK1 protein was isolated by two cycles of immunoprecipitation using anti-amino terminus MKK1 antibody. The proteins were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography.

The retroviral construct used (pSR/MSV-Tkneo) was previously described (Mol. Cell. Biol. 11:1785-1792 (1991)). The MKK1 sense construct represents the full length gene lacking the poly-adenylation sequences. The MKK1 antisense construct represents the 5′ fragment EcoRI-PvuII cloned in the reverse orientation. Both the sense and antisense constructs are driven by the retroviral long terminal repeat (LTR).

The results of the experiment, as shown in FIGS. 6A-6B, indicate that expression of the MKK1 anti-sense sequences in the cultured bone marrow elements is associated with decreased expression of MKK1 and decreased levels of AChE, an indicator of megakaryocyte growth and differentiation.

10. EXAMPLE

MXX1 Protein Tyrosine Kinase Activity

The protein tyrosine kinase activity of MKK1 was demonstrated through the incorporation of ³²P in poly (Glu-Tyr) substrate by MKK1. The MKK1 used to demonstrate protein tyrosine kinase activity was obtained from 293 cells transiently overexpressing MKK1.

10.1. Materials and Methods

293 cells were transiently transfected with pCMP1-MKK1 (White, et al. J. Biol. Chem. (1987) 263:2969-2980), an expression vector containing nucleic acid encoding MKK1 operably linked to the CMV promoter. The 293 cells were harvested 48 hours after transfection and lysed in 1 ml of lysis buffer (20 mM tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1 mM sodium orthovanadate, 0.1 mM PMSF) per one 10 cm culture dish. The lysates were collected by centrifugation at 20,000 g for 30 minutes. Control lysates were prepared in the same way as non-transfected 293 cells.

One ml aliquotes of the MKK1 transfected cell lysates and the control lysates, respectively, were incubated under constant agitation for 2 hours at 4° C. with 5 μl of polyclonal antiserum raised against a GST fusion protein containing amino acid residues 25-223 of the N-terminus of MKK1 and 15 μl of Protein A-Sepharose. The resultant MKK1 immunoprecipitates were spun down and washed with the lysis buffer twice; 50 mM tris-HCl [pH 7.4], twice; and either Mg²⁺- or Mn²⁺-containing kinase buffers (50 mM tris-HCl [pH 7.4], 5 mM of MgCl₂ or 5 mM MnCl₂, respectively), once. After the washes, the MKK1 immunoprecipitates were resuspended in 40 μl of either the Mg²⁺- or Mn²⁺-containing kinase buffers containing 1 mg/ml of poly (Glu-Tyr) 4:1 (Sigma, P-0275) and 10 μCi of gamma-32P-ATP (Amersham) and incubated for 20 minutes at 37° C. 10 μl aliquotes of the kinase reactions were spotted on glass paper filter (1205 Betaplate cassete filtermat, Beckman), and the filters were washed for 1.5 hours in three, sequential 100 ml volumes of (10% trichloroacetic acid, 10 mM sodium pyrophosphate). The washed filters were dried and analyzed by scintillation counting using BS Betaplate liquid scintillation counter (Beckman).

10.2. Results

Immunoprecipitates from MKK1 transiently transfected cells exhibited a 20-50-fold increase in counts over control cells, while no significant increase in counts was detected for immunoprecipitates from non-transfected cells or for MKK1 kinase assays carried out in the absence of poly (Glu-Tyr). Higher tyrosine kinase activity of MKK1 was observed in the presence of manganese than in the presence of magnesium.

11. EXAMPLE

Biological Activity of MKK1

In order to assess the biological activity of MKK1, a murine megakaryocytic cell line (L-8057) was infected with a retrovirus containing nucleic acid encoding MKK1 and the selectable drug-resistance marker, neomycin. Cell growth of the MKK1 expressing L-8057 cell lines and the ability of MKK1 expressing L-8057 cell lines to induce differentiation in the presence of tetradecanoyl phorbol acetate (TPA) was determined. The MKK1 expressing L-8057 cell lines were incubated with various cytokines to determine if any cytokine had the ability to block growth inhibition by MKK1.

11.1. Materials and Methods

11.1.1. Retroviral Infection of L-8057 cells

Aliquotes of about 2×10⁶ L-8057 cells (L-8057 cells were obtained from Dr. Yoji Ishida at the Iwate Medical University, Morioka, Japan) (about 1.5 ml of 8×10⁵ cells per ml in 15 ml conical tube) were prepared and collected by centrifugation. The collected cells were resuspended in 1.5 ml of viral stock of either ψ⁻ Eco msvTKneo MKK1 full length, designated V25, or ψ⁻ Eco msvTKneo, designated V6, (pPSR-a described in Muller et al.(1992) Mol. Cell. Bio. 11:1785-1792) containing 1.5 μl per ml of 6 mg/ml polybrene (Sigma, H 9268) and incubated at 37° C./5% CO₂ for 3 hours, with swirling every 30 minutes. The cells were then collected by centrifugation. An additional 1.5 ml of viral stock containing polybrene was added and the cells were incubated for an additional 3 hours as described above. The cells were collected by centrifugation, resuspended in 2 ml of standard L-8057 medium (20% fetal bovine serum (FBS), 40% RPMI, 40% IMDM (Gibco)), placed in 6 well plates and incubated at 37° C. for 2 days.

The cells were collected by centrifugation, resuspended in 1 ml of medium containing 1 mg/ml G-418 and placed in 6 cm dishes with 5 ml of medium containing G-418.

Non-infected L-8057 cells grown in media containing G-418 (1×10⁵ cells per ml in 10 cm dish) were used as a control.

Following drug-selection in G-418 and cell expansion, cells counts were made on days 5 and 6. Expression of MKK1 was verified by Western Blot.

11.1.2. Cell Growth Measurement of MKK1 Expressing L-8057 Cells

Cell growth of the MKK1 expressing L-8057 cell lines, designated V25A and V25B, along with mock-infected L-8057 cells and control cells was measured. Duplicate 6 well dishes plated at a cell density of 1×10⁵/ml or 3.3×10⁴/ml cells were prepared in 1% Nutridoma (Boehringer Mannheim). Cell counts were taken at days 5 and 6.

vol. 2 × 10⁵ Day 5 cell counts: Day 6 cell counts: cells CNTL 1.9 × 10⁶/mL 1.6 × 10⁶/mL 0.125 ml V6 5.2 × 10⁵/mL 4.0 × 10⁵/mL  0.5 ml V25A 0.8 × 10⁵/mL 0.8 × 10⁵/mL  2.5 ml V25B 1.6 × 10⁵/mL 1.0 × 10⁵/mL  2.0 ml

11.1.3. Growth Factor Response of MKK1 Expressing L-8057 Cells

The cells from one dish each of L-8057 cells infected with a retrovirus containing full length MKK1, designated V25A, and empty vector, designated V⁶⁻¹ were collected through centrifugation, washed with 10 ml of (IMDM, 1% Nutridoma, glutamine (Gibco), penicillin-streptomycin (Gibco) recentrifuged and resuspended in 2 ml of same medium. The cells were diluted to 2×10⁵ cells/ml and 50 μl (or 10⁴ cells) were added per well of a 96 well plate and incubated for 24 hours under conditions of serum starvation. After serum starvation, rat stem cell factor, cKit ligand, IL-3, IL-6, IL-11, IL-1β, EPO, human MPL ligand, Spleen Cell Conditioned Medium (IL-3, IL-6) to a volume of 10%, WEHI 38 Conditioned Medium (IL-3, GM-CSF) to a volume of 10%, and fetal bovine serum to a volume of 10% (as a positive control) were added to the cells. The cells were allowed to grow for three days and cell growth was measured in a standard MTT (tetrazolium) assay (Mosmann, J. Imm. Meth. (1983) 65:55-63)

11.2. Results

11.2.1. Cell Growth

The highest MKK1 expression was observed in cells designated L-8057 V25A. No expression was observed in cells infected with the empty vector control, designated L-8057 V6. The growth rate was then examined under growth limiting conditions, i.e., in serum-free media. Compared with the empty vector control, cells infected with MKK1 grew at a much slower rate, see FIG. 12.

11.2.2. Cytokine Stimulation

Stimulation of the MKK1 infected L-8057 cells with a panel of cytokines, including rat stem cell factor (SCF), IL-3, IL-6, IL-11, IL-1β and EPO, revealed that only rat stem cell factor (SCF) was capable of overcoming the inhibition of proliferation produced by the hyperexpression of MKK1, see FIG. 13. The control cells also responded to SCF. The percent increase in the growth of the control cells treated with rat SCF and the MKK1 infected cells treated with rat SCF was similar. The data suggest that rat stem cell factor does not have the same signalling transduction pathway as MKK1.

11.2.3. Cell Differentiation

In order to test the effect of MKK1 on megakaryocyte differentiation, a murine megakaryocytic cell line (L-8057) was engineered to express MKK1, as described in Section 11.1.1. Induction of polyploidy (cells with DNA content greater than 4N) is a hallmark of megakaryocytic differentiation. L-8057 can be induced to differentiate (measured by induction of polyploidy and expression of the enzyme acetylcholinasterase in murine systems) by treatment with tetradecanoyl phorbol acetate (TPA) (Ishida et al., Exp. Hematol. 21:289-298, 1993). FIG. 14 demonstrates the effect of TPA on either control cells or cells that express MKK1. Both control cells and MKK1 expressing cells became polyploid to the same extent in response to treatment with TPA for 3 days. This data suggest that induction of differentiation is not affected by the presence of MKK1.

Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. It is also to be understood that all base pair sizes given for nucleotides are approximate and are used for purposes of description.

All references cited herein are hereby incorporated by reference in their entirety.

21 2000 base pairs nucleic acid unknown unknown not provided 1 CTCGCTCCAA GTTGTGCAGC CGGGACCGCC TCGGGGTGTG CAGCCGGCTC GCGGAGGCCC 60 TCCTGGGGGC GGGCGCGGGG CGGCTCGGGG GCGCCCCCTG AGCAGAAAAC AGGAAGAACC 120 AGGCTCGGTC CAGTGGCACC CAGCTCCCTA CCTCCTGTGC CAGCCGCCTG GCCTGTGGCA 180 GGCCATTCCC AGCGTCCCCG ACTGTGACCA CTTGCTCAGT GTGCCTCTCA CCTGCCTCAG 240 TTTCCCTCTG GGGGGCGATG GCGGGGCGAG GCTCTCTGGT TTCCTGGCGG GCATTTCACG 300 GCTGTGATTC TGCTGAGGAA CTTCCCCGGG TGAGCCCCCG CTTCCTCCGA GCCTGGCACC 360 CCCCTCCCGT CTCAGCCAGG ATGCCAACGA GGCGCTGGGC CCCGGGCACC CAGTGTATCA 420 CCAAATGCGA GCACACCCGC CCCAAGCCAG GGGAGCTGGC CTTCCGCAAG GGCGACGTGG 480 TCACCATCCT GGAGGCCTGC GAGAACAAGA GCTGGTACCG CGTCAAGCAC CACACCAGTG 540 GACAGGAGGG GCTGCTGGCA GCTGGGGCGC TGCGGGAGCG GGAGGCCCTC TCCGCAGACC 600 CCAAGCTCAG CCTCATGCCG TGGTTCCACG GGAAGATCTC GGGCCAGGAG GCTGTCCAGC 660 AGCTGCAGCC TCCCGAGGAT GGGCTGTTCC TGGTGCGGGA GTCCGCGCGC CACCCCGGCG 720 ACTACGTCCT GTGCGTGAGC TTTGGCCGCG ACGTCATCCA CTACCGCGTG CTGCACCGCG 780 ACGGCCACCT CACAATCGAT GAGGCCGTGT TCTTCTGCAA CCTCATGGAC ATGGTGGAGC 840 ATTACAGCAA GGACAAGGGC GCTATCTGCA CCAAGCTGGT GAGACCAAAG CGGAAACACG 900 GGACCAAGTC GGCCGAGGAG GAGCTGGCCA GGGCGGGCTG GTTACTGAAC CTGCAGCATT 960 TGACATTGGG AGCACAGATC GGAGAGGGAG AGTTTGGAGC TGTCCTGCAG GGTGAGTACC 1020 TGGGGCAAAA GGTGGCCGTG AAGAATATCA AGTGTGATGT GACAGCCCAG GCCTTCCTGG 1080 ACGAGACGGC CGTCATGACG AAGATGCAAC ACGAGAACCT GGTGCGTCTC CTGGGCGTGA 1140 TCCTGCACCA GGGGCTGTAC ATTGTCATGG AGCACGTGAG CAAGGGCAAC CTGGTGAACT 1200 TTCTGCGGAC CCGGGGTCGA GCCCTCGTGA ACACCGCTCA GCTCCTGCAG TTTTCTCTGC 1260 ACGTGGCCGA GGGCATGGAG TACCTGGAGA GCAAGAAGCT TGTGCACCGC GACCTGGCCG 1320 CCCGCAACAT CCTGGTCTCA GAGGACCTGG TGGCCAAGGT CAGCGACTTT GGCCTGGCCA 1380 AAGCCGAGCG GAAGGGGCTA GACTCAAGCC GGCTGCCCGT CAAGTGGACG GCGCCCGAGG 1440 CTCTCAAACA CGGGAAGTTC ACCAGCAAGT CGGATGTCTG GAGTTTTGGG GTGCTGCTCT 1500 GGGAGGTCTT CTCATATGGA CGGGCTCCGT ACCCTAAAAT GTCACTGAAA GAGGTGTCGG 1560 AGGCCGTGGA GAAGGGGTAC CGCATGGAAC CCCCCGAGGG CTGTCCAGGC CCCGTGCACG 1620 TCCTCATGAG CAGCTGCTGG GAGGCAGAGC CCGCCCGCCG GCCACCCTTC CGCAAACTGG 1680 CCGAGAAGCT GGCCCGGGAG CTACGCAGTG CAGGTGCCCC AGCCTCCGTC TCAGGGCAGG 1740 ACGCCGACGG CTCCACCTCG CCCCGAAGCC AGGAGCCCTG ACCCCACCCG GTGGGGCCCT 1800 TGGCCCCAGA GGACCGAGAG AGTGGAGAGT GCGGCGTGGG GGCACTGACC AGGCCCAAGG 1860 AGGGTCCAGG CGGGCAAGTC ATCCTCCTGG TGCCCACAGC AGGGGCTGGC CCACGTAGGG 1920 GGCTCTGGGC GGCCCGTGGA CACCCCAGAC CTGCGAAGGA TGATCGCCCG ATAAAGACGG 1980 ATTCTAAGGA CTCTAAAAAA 2000 507 amino acids amino acid unknown unknown None not provided 2 Met Ala Gly Arg Gly Ser Leu Val Ser Trp Arg Ala Phe His Gly Cys 1 5 10 15 Asp Ser Ala Glu Glu Leu Pro Arg Val Ser Pro Arg Phe Leu Arg Ala 20 25 30 Trp His Pro Pro Pro Val Ser Ala Arg Met Pro Thr Arg Arg Trp Ala 35 40 45 Pro Gly Thr Gln Cys Ile Thr Lys Cys Glu His Thr Arg Pro Lys Pro 50 55 60 Gly Glu Leu Ala Phe Arg Lys Gly Asp Val Val Thr Ile Leu Glu Ala 65 70 75 80 Cys Glu Asn Lys Ser Trp Tyr Arg Val Lys His His Thr Ser Gly Gln 85 90 95 Glu Gly Leu Leu Ala Ala Gly Ala Leu Arg Glu Arg Glu Ala Leu Ser 100 105 110 Ala Asp Pro Lys Leu Ser Leu Met Pro Trp Phe His Gly Lys Ile Ser 115 120 125 Gly Gln Glu Ala Val Gln Gln Leu Gln Pro Pro Glu Asp Gly Leu Phe 130 135 140 Leu Val Arg Glu Ser Ala Arg His Pro Gly Asp Tyr Val Leu Cys Val 145 150 155 160 Ser Phe Gly Arg Asp Val Ile His Tyr Arg Val Leu His Arg Asp Gly 165 170 175 His Leu Thr Ile Asp Glu Ala Val Phe Phe Cys Asn Leu Met Asp Met 180 185 190 Val Glu His Tyr Ser Lys Asp Lys Gly Ala Ile Cys Thr Lys Leu Val 195 200 205 Arg Pro Lys Arg Lys His Gly Thr Lys Ser Ala Glu Glu Glu Leu Ala 210 215 220 Arg Ala Gly Trp Leu Leu Asn Leu Gln His Leu Thr Leu Gly Ala Gln 225 230 235 240 Ile Gly Glu Gly Glu Phe Gly Ala Val Leu Gln Gly Glu Tyr Leu Gly 245 250 255 Gln Lys Val Ala Val Lys Asn Ile Lys Cys Asp Val Thr Ala Gln Ala 260 265 270 Phe Leu Asp Glu Thr Ala Val Met Thr Lys Met Gln His Glu Asn Leu 275 280 285 Val Arg Leu Leu Gly Val Ile Leu His Gln Gly Leu Tyr Ile Val Met 290 295 300 Glu His Val Ser Lys Gly Asn Leu Val Asn Phe Leu Arg Thr Arg Gly 305 310 315 320 Arg Ala Leu Val Asn Thr Ala Gln Leu Leu Gln Phe Ser Leu His Val 325 330 335 Ala Glu Gly Met Glu Tyr Leu Glu Ser Lys Lys Leu Val His Arg Asp 340 345 350 Leu Ala Ala Arg Asn Ile Leu Val Ser Glu Asp Leu Val Ala Lys Val 355 360 365 Ser Asp Phe Gly Leu Ala Lys Ala Glu Arg Lys Gly Leu Asp Ser Ser 370 375 380 Arg Leu Pro Val Lys Trp Thr Ala Pro Glu Ala Leu Lys His Gly Lys 385 390 395 400 Phe Thr Ser Lys Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu 405 410 415 Val Phe Ser Tyr Gly Arg Ala Pro Tyr Pro Lys Met Ser Leu Lys Glu 420 425 430 Val Ser Glu Ala Val Glu Lys Gly Tyr Arg Met Glu Pro Pro Glu Gly 435 440 445 Cys Pro Gly Pro Val His Val Leu Met Ser Ser Cys Trp Glu Ala Glu 450 455 460 Pro Ala Arg Arg Pro Pro Phe Arg Lys Leu Ala Glu Lys Leu Ala Arg 465 470 475 480 Glu Leu Arg Ser Ala Gly Ala Pro Ala Ser Val Ser Gly Gln Asp Ala 485 490 495 Asp Gly Ser Thr Ser Pro Arg Ser Gln Glu Pro 500 505 2500 base pairs nucleic acid unknown unknown not provided 3 CCGCTTTTTG CTTAGAGCTT GAGAGTCAAA GTTAAGGACC CACATGTATA CTTCGGCTCT 60 AGCGAGTCTA AGGATGATAA TATGGATACA AAATCTATTC TAGAAGAACT TCTTCTCAAA 120 AGATCACAGC AAAAGAAGAA AATGTCACCA AATAATTACA AAGAACGGCT TTTTGTTTTG 180 ACCAAAACAA ACCTTTCCTA CTATGAATAT GACAAAATGA AAAGGGGCAG CAGAAAAGGA 240 TCCATTGAAA TTAAGAAAAT CAGATGTGTG GAGAAAGTAA ATCTCGAGGA GCAGACGCCT 300 GTAGAGAGAC AGTACCCATT TCAGATTGTC TATAAAGATG GGCTTCTCTA TGTCTATGCA 360 TCAAATGAAG AGAGCCGAAG TCAGTGGTTG AAAGCATTAC AAAAAGAGAT AAGGGGTAAC 420 CCCCACCTGC TGGTCAAGTA CCATAGTGGG TTCTTCGTGG ACGGGAAGTT CCTGTGTTGC 480 CAGCAGAGCT GTAAAGCAGC CCCAGGATGT ACCCTCTGGG AAGCATATGC TAATCTGCAT 540 ACTGCAGTCA ATGAAGAGAA ACACAGAGTT CCCACCTTCC CAGACAGAGT GCTGAAGATA 600 CCTCGGGCAG TTCCTGTTCT CAAAATGGAT GCACCATCTT CAAGTACCAC TCTAGCCCAA 660 TATGACAACG AATCAAAGAA AAACTATGGC TCCCAGCCAC CATCTTCAAG TACCAGTCTA 720 GCGCAATATG ACAGCAACTC AAAGAAAATC TATGGCTCCC AGCCAAACTT CAACATGCAG 780 TATATTCCAA GGGAAGACTT CCCTGACTGG TGGCAAGTAA GAAAACTGAA AAGTAGCAGC 840 AGCAGTGAAG ATGTTGCAAG CAGTAACCAA AAAGAAAGAA ATGTGAATCA CACCACCTCA 900 AAGATTTCAT GGGAATTCCC TGAGTCAAGT TCATCTGAAG AAGAGGAAAA CCTGGATGAT 960 TATGACTGGT TTGCTGGTAA CATCTCCAGA TCACAATCTG AACAGTTACT CAGACAAAAG 1020 GGAAAAGAAG GAGCATTTAT GGTTAGAAAT TCGAGCCAAG TGGGAATGTA CACAGTGTCC 1080 TTATTTAGTA AGGCTGTGAA TGATAAAAAA GGAACTGTCA AACATTACCA CGTGCATACA 1140 AATGCTGAGA ACAAATTATA CCTGGCAGAA AACTACTGTT TTGATTCCAT TCCAAAGCTT 1200 ATTCATTATC ATCAACACAA TTCAGCAGGC ATGATCACAC GGCTCCGCCA CCCTGTGTCA 1260 ACAAAGGCCA ACAAGGTCCC CGACTCTGTG TCCCTGGGAA ATGGAATCTG GGAACTGAAA 1320 AGAGAAGAGA TTACCTTGTT GAAGGAGCTG GGAAGTGGCC AGTTTGGAGT GGTCCAGCTG 1380 GGCAAGTGGA AGGGGCAGTA TGATGTTGCT GTTAAGATGA TCAAGGAGGG CTCCATGTCA 1440 GAAGATGAAT TCTTTCAGGA GGCCCAGACT ATGATGAAAC TCAGCCATCC CAAGCTGGTT 1500 AAATTCTATG GAGTGTGTTC AAAGGAATAC CCCATATACA TAGTGACTGA ATATATAAGC 1560 AATGGCTGCT TGCTGAATTA CCTGAGGAGT CACGGAAAAG GACTTGAACC TTCCCAGCTC 1620 TTAGAAATGT GCTACGATGT CTGTGAAGGC ATGGCCTTCT TGGAGAGTCA CCAATTCATA 1680 CACCGGGACT TGGCTGCTCG TAACTGCTTG GTGGACAGAG ATCTCTGTGT GAAAGTATCT 1740 GACTTTGGAA TGACAAGGTA TGTTCTTGAT GACCAGTATG TCAGTTCAGT CGGAACAAAG 1800 TTTCCAGTCA AGTGGTCAGC TCCAGAGGTG TTTCATTACT TCAAATACAG CAGCAAGTCA 1860 GACGTATGGG CATTTGGGAT CCTGATGTGG GAGGTGTTCA GCCTGGGGAA GCAGCCCTAT 1920 GACTTGTATG ACAACTCCCA GGTGGTTCTG AAGGTCTCCC AGGGCCACAG GCTTTACCGG 1980 CCCCACCTGG CATCGGACAC CATCTACCAG ATCATGTACA GCTGCTGGCA CGAGCTTCCA 2040 GAAAAGCGTC CCACATTTCA GCAACTCCTG TCTTCCATTG AACCACTTCG GGAAAAAGAC 2100 AAGCATTGAA GAAGAAATTA GGAGTGCTGA TAAGAATGAA TATAGATGCT GGCCAGCATT 2160 TTCATTCATT TTAAGGAAAG TAGCAAGGCA TAATGTAATT TAGCTAGTTT TTAATAGTGT 2220 TCTCTGTATT GTCTATTATT TAGAAATGAA CAAGGCAGGA AACAAAAGAT TCCCTTGAAA 2280 TTTAGGTCAA ATTAGTAATT TTGTTTATGC TGCCCCTGAT ATAACACTTT CCAGCCTATA 2340 GCAGAAGCAC ATTTTCAGAC TGCAATATAG AGACTGTGTT CATGTGTAAA GACTGAGCAG 2400 AACTGAAAAA TTACTTATTG GATATTCATT CTTTTCTTTA TATTGTCATT GTCACAACAA 2460 TTAAATATAC TACCAAGTAC AAAAAAAAAA AAAAAAAAAA 2500 675 amino acids amino acid unknown unknown None not provided 4 Met Asp Thr Lys Ser Ile Leu Glu Glu Leu Leu Leu Lys Arg Ser Gln 1 5 10 15 Gln Lys Lys Lys Met Ser Pro Asn Asn Tyr Lys Glu Arg Leu Phe Val 20 25 30 Leu Thr Lys Thr Asn Leu Ser Tyr Tyr Glu Tyr Asp Lys Met Lys Arg 35 40 45 Gly Ser Arg Lys Gly Ser Ile Glu Ile Lys Lys Ile Arg Cys Val Glu 50 55 60 Lys Val Asn Leu Glu Glu Gln Thr Pro Val Glu Arg Gln Tyr Pro Phe 65 70 75 80 Gln Ile Val Tyr Lys Asp Gly Leu Leu Tyr Val Tyr Ala Ser Asn Glu 85 90 95 Glu Ser Arg Ser Gln Trp Leu Lys Ala Leu Gln Lys Glu Ile Arg Gly 100 105 110 Asn Pro His Leu Leu Val Lys Tyr His Ser Gly Phe Phe Val Asp Gly 115 120 125 Lys Phe Leu Cys Cys Gln Gln Ser Cys Lys Ala Ala Pro Gly Cys Thr 130 135 140 Leu Trp Glu Ala Tyr Ala Asn Leu His Thr Ala Val Asn Glu Glu Lys 145 150 155 160 His Arg Val Pro Thr Phe Pro Asp Arg Val Leu Lys Ile Pro Arg Ala 165 170 175 Val Pro Val Leu Lys Met Asp Ala Pro Ser Ser Ser Thr Thr Leu Ala 180 185 190 Gln Tyr Asp Asn Glu Ser Lys Lys Asn Tyr Gly Ser Gln Pro Pro Ser 195 200 205 Ser Ser Thr Ser Leu Ala Gln Tyr Asp Ser Asn Ser Lys Lys Ile Tyr 210 215 220 Gly Ser Gln Pro Asn Phe Asn Met Gln Tyr Ile Pro Arg Glu Asp Phe 225 230 235 240 Pro Asp Trp Trp Gln Val Arg Lys Leu Lys Ser Ser Ser Ser Ser Glu 245 250 255 Asp Val Ala Ser Ser Asn Gln Lys Glu Arg Asn Val Asn His Thr Thr 260 265 270 Ser Lys Ile Ser Trp Glu Phe Pro Glu Ser Ser Ser Ser Glu Glu Glu 275 280 285 Glu Asn Leu Asp Asp Tyr Asp Trp Phe Ala Gly Asn Ile Ser Arg Ser 290 295 300 Gln Ser Glu Gln Leu Leu Arg Gln Lys Gly Lys Glu Gly Ala Phe Met 305 310 315 320 Val Arg Asn Ser Ser Gln Val Gly Met Tyr Thr Val Ser Leu Phe Ser 325 330 335 Lys Ala Val Asn Asp Lys Lys Gly Thr Val Lys His Tyr His Val His 340 345 350 Thr Asn Ala Glu Asn Lys Leu Tyr Leu Ala Glu Asn Tyr Cys Phe Asp 355 360 365 Ser Ile Pro Lys Leu Ile His Tyr His Gln His Asn Ser Ala Gly Met 370 375 380 Ile Thr Arg Leu Arg His Pro Val Ser Thr Lys Ala Asn Lys Val Pro 385 390 395 400 Asp Ser Val Ser Leu Gly Asn Gly Ile Trp Glu Leu Lys Arg Glu Glu 405 410 415 Ile Thr Leu Leu Lys Glu Leu Gly Ser Gly Gln Phe Gly Val Val Gln 420 425 430 Leu Gly Lys Trp Lys Gly Gln Tyr Asp Val Ala Val Lys Met Ile Lys 435 440 445 Glu Gly Ser Met Ser Glu Asp Glu Phe Phe Gln Glu Ala Gln Thr Met 450 455 460 Met Lys Leu Ser His Pro Lys Leu Val Lys Phe Tyr Gly Val Cys Ser 465 470 475 480 Lys Glu Tyr Pro Ile Tyr Ile Val Thr Glu Tyr Ile Ser Asn Gly Cys 485 490 495 Leu Leu Asn Tyr Leu Arg Ser His Gly Lys Gly Leu Glu Pro Ser Gln 500 505 510 Leu Leu Glu Met Cys Tyr Asp Val Cys Glu Gly Met Ala Phe Leu Glu 515 520 525 Ser His Gln Phe Ile His Arg Asp Leu Ala Ala Arg Asn Cys Leu Val 530 535 540 Asp Arg Asp Leu Cys Val Lys Val Ser Asp Phe Gly Met Thr Arg Tyr 545 550 555 560 Val Leu Asp Asp Gln Tyr Val Ser Ser Val Gly Thr Lys Phe Pro Val 565 570 575 Lys Trp Ser Ala Pro Glu Val Phe His Tyr Phe Lys Tyr Ser Ser Lys 580 585 590 Ser Asp Val Trp Ala Phe Gly Ile Leu Met Trp Glu Val Phe Ser Leu 595 600 605 Gly Lys Gln Pro Tyr Asp Leu Tyr Asp Asn Ser Gln Val Val Leu Lys 610 615 620 Val Ser Gln Gly His Arg Leu Tyr Arg Pro His Leu Ala Ser Asp Thr 625 630 635 640 Ile Tyr Gln Ile Met Tyr Ser Cys Trp His Glu Leu Pro Glu Lys Arg 645 650 655 Pro Thr Phe Gln Gln Leu Leu Ser Ser Ile Glu Pro Leu Arg Glu Lys 660 665 670 Asp Lys His 675 2770 base pairs nucleic acid unknown unknown not provided 5 CCGGACTGGT CGAAAGACAG GAACAGACTT GAAACAGGGG GAGAGCTCCT GGCGAAACGA 60 AGACGTGGAG GTTTTACCAG GGATAAGAAG AAAAGACACC TTCCTAGTGA GCAGCTGCCC 120 AGCTCCTGCT CAGTTTTGCC TCGGGGTAGC ACCTCCAGCC ACAGAAAGCA AGCCGGTAAG 180 TCTCTCCAGG TAGGACTTGC TGCAACCCAG CTGCTGGACT GATCTGAAAC GGGACTTTGC 240 ATACTCTCCG AAGTATGGTG AGTTGGTGCT GACTTCAAAG TTGCCTGGTG AAGGAAGATA 300 AGGTGGATCG CAGAGACTAA GGGGAGAGGG AGAAGCCCTG CTCCTCTTCT CCCCACCAAG 360 GCACAATGAG CAACATCTGT CAGAGGCTCT GGGAGTACCT AGAACCCTAT CTCCCCTGTT 420 TGTCCACGGA GGCAGACAAG TCAACCGTGA TTGAAAATCC AGGGGCCCTT TGCTCTCCCC 480 AGTCACAGAG GCATGGCCAC TACTTTGTGG CTTTGTTTGA TTACCAGGCT CGGACTGCTG 540 AGGACTTGAG CTTCCGAGCA GGTGACAAAC TTCAAGTTCT GGACACTTTG CATGAGGGCT 600 GGTGGTTTGC CAGACACTTG GAGAAAAGAC GAGATGGCTC CAGTCAGCAA CTACAAGGCT 660 ATATTCCTTC TAACTACGTG GCTGAGGACA GAAGCCTACA GGCAGAGCCG TGGTTCTTTG 720 GAGCAATCGG AAGATCAGAT GCAGAGAAAC AACTATTATA TTCAGAAAAC AAGACCGGTT 780 CCTTTCTAAT CAGAGAAAGT GAAAGCCAAA AAGGAGAATT CTCTCTTTCA GTTTTAGATG 840 GAGCAGTTGT AAAACACTAC AGAATTAAAA GACTGGATGA AGGGGGATTT TTTCTCACGC 900 GAAGAAGAAT CTTTTCAACA CTGAACGAAT TTGTGAGCCA CTACACCAAG ACAAGTGACG 960 GCCTGTGTGT CAAGCTGGGG AAACCATGCT TAAAGATCCA GGTCCCAGCT CCATTTGATT 1020 TGTCGTATAA AACCGTGGAC CAATGGGAGA TAGACCGCAA CTCCATACAG CTTCTGAAGC 1080 GATTGGGATC TGGTCAGTTT GGCGAAGTAT GGGAAGGTCT GTGGAACAAT ACCACTCCAG 1140 TAGCAGTGAA AACATTAAAA CCAGGTTCAA TGGATCCAAA TGACTTCCTG AGGGAGGCAC 1200 AGATAATGAA GAACCTAAGA CATCCAAAGC TTATCCAGCT TTATGCTGTT TGCACTTTAG 1260 AAGATCCAAT TTATATTATT ACAGAGTTGA TGAGACATGG AAGTCTGCAA GAATATCTCC 1320 AAAATGACAC TGGATCAAAA ATCCATCTGA CTCAACAGGT AGACATGGCG GCACAGGTTG 1380 CCTCTGGAAT GGCCTATCTG GAGTCTCGGA ACTACATTCA CAGAGATCTG GCTGCCAGAA 1440 ATGTCCTCGT TGGTGAACAT AATATCTACA AAGTAGCAGA TTTTGGACTT GCCAGAGTTT 1500 TTAAGGTAGA TAATGAAGAC ATCTATGAAT CTAGACACGA AATAAAGCTG CCGGTGAAGT 1560 GGACTGCGCC CGAAGCCATT CGTAGTAATA AATTCAGCAT TAAGTCCGAT GTATGGTCAT 1620 TTGGAATCCT TCTTTATGAA ATCATTACTT ATGGCAAAAT GCCTTACAGT GGTATGACAG 1680 GTGCCCAGGT AATCCAGATG TTGGCTCAAA ACTATAGACT TCCGCAACCA TCCAACTGTC 1740 CACAGCAATT TTACAACATC ATGTTGGAGT GCTGGAATGC AGAGCCTAAG GAACGACCTA 1800 CATTTGAGAC ACTGCGTTGG AAACTTGAAG ACTATTTTGA AACAGACTCT TCATATTCAG 1860 ATGCAAATAA CTTCATAAGA TGAACACTGG AGAAGAATAT CAAATAATAA AGTAGCAAAA 1920 CAAATTCAAA TAATCCATTC CAAAATACAA TGTTATCAAC CAACTGCACA ATCAGTTTAT 1980 CCTGACATAT TCAAGTGATA GGATAAAGTT GGCCATGTAT TATGAAAAAG ATTATTTGTG 2040 CATTTTATTG ACTGGGCAAC ACTGCAGGAC AGTCAAGGTC ATATATAATT GCTCACTGCC 2100 TGGAAAATTA AGCACACTAA ACCAAGTTAT TTTTCTTTTT AAGAGATACT TACATTTCCA 2160 TTTATTGTTT GAAATGTCGC GATCAAGAGA ATCAACAGAT GATAGTCCAA TTTTTACTCA 2220 GTGATGACTG TGTAGCATTT TCCTGTTTAC TGATTAGAGT GGTTATTCAT TATTCCTCAG 2280 ATTGCTGAAT CCCATCAGGC TGTTATTATG AAGGAATTTG ATTGCTTTGC TGCACAGCAG 2340 GACCTGTGCT TTGAGATTTT TTTTTCTCTT TTAAAATATC CTGTAACTAC AATGATGGTA 2400 AAGCCATGTT AAATGACTTG ATTGTACTTG GAGTAATTGC ACATTTTTTT CTATGCATAA 2460 AAAAATGATG CAGCTGTTGA GAAAACGAAG TCTTTTTCAT TTTGCAGAAG GAAATGATGG 2520 AATTTTTCTG TACTTCAGTA TGTGTCAACT GAGAGTCATA TACATTAGTT TTAATCTCTT 2580 AATATTGAGA ATCAGGTTGC AAAACGGATG AGTTATTATC TATGGAAATG TGAGAAATGT 2640 CTAATAGCCC ATAAAGTCTG AGAAATAGGT ATCAAAATAG TTTAGGAAAA TGAGAGGAGA 2700 ACAGTAGGAT TGCTGTGGCC TAGACTTCTG AGTAATTAAT AAAGAAAAAG AAGTACCAAA 2760 AAAAAAAAAA 2770 506 amino acids amino acid unknown unknown None not provided 6 Met Ser Asn Ile Cys Gln Arg Leu Trp Glu Tyr Leu Glu Pro Tyr Leu 1 5 10 15 Pro Cys Leu Ser Thr Glu Ala Asp Lys Ser Thr Val Ile Glu Asn Pro 20 25 30 Gly Ala Leu Cys Ser Pro Gln Ser Gln Arg His Gly His Tyr Phe Val 35 40 45 Ala Leu Phe Asp Tyr Gln Ala Arg Thr Ala Glu Asp Leu Ser Phe Arg 50 55 60 Ala Gly Asp Lys Lys Leu Gln Val Leu Asp Thr Leu His Glu Gly Trp 65 70 75 80 Trp Phe Ala Arg His Leu Glu Lys Arg Arg Asp Gly Ser Ser Gln Gln 85 90 95 Leu Gln Gly Tyr Ile Pro Ser Asn Tyr Val Ala Glu Asp Arg Ser Leu 100 105 110 Gln Ala Glu Pro Trp Phe Phe Gly Ala Ile Gly Arg Ser Asp Ala Glu 115 120 125 Lys Gln Leu Leu Tyr Ser Glu Asn Lys Thr Gly Ser Phe Leu Ile Arg 130 135 140 Glu Ser Glu Ser Gln Lys Gly Glu Phe Ser Leu Ser Val Leu Asp Gly 145 150 155 160 Ala Val Val Lys His Tyr Arg Ile Lys Arg Leu Asp Glu Gly Gly Phe 165 170 175 Phe Leu Thr Arg Arg Arg Ile Phe Ser Thr Leu Asn Glu Phe Val Ser 180 185 190 His Tyr Thr Lys Thr Ser Asp Gly Leu Cys Val Lys Leu Gly Lys Pro 195 200 205 Cys Leu Lys Ile Gln Val Pro Ala Pro Phe Asp Leu Ser Tyr Lys Thr 210 215 220 Val Asp Gln Trp Glu Ile Asp Arg Asn Ser Ile Gln Leu Leu Lys Arg 225 230 235 240 Leu Gly Ser Gly Gln Phe Gly Glu Val Trp Glu Gly Leu Trp Asn Asn 245 250 255 Thr Thr Pro Val Ala Val Lys Thr Leu Lys Pro Gly Ser Met Asp Pro 260 265 270 Asn Asp Phe Leu Arg Glu Ala Gln Ile Met Lys Asn Leu Arg His Pro 275 280 285 Lys Leu Ile Gln Leu Tyr Ala Val Cys Thr Leu Glu Asp Pro Ile Tyr 290 295 300 Ile Ile Thr Glu Leu Met Arg His Gly Ser Leu Gln Glu Tyr Leu Gln 305 310 315 320 Asn Asp Thr Gly Ser Lys Ile His Leu Thr Gln Gln Tyr Asp Met Ala 325 330 335 Ala Gln Val Ala Ser Gly Met Ala Tyr Leu Glu Ser Arg Asn Tyr Ile 340 345 350 His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gly Glu His Asn Ile 355 360 365 Tyr Lys Val Ala Asp Phe Gly Leu Ala Arg Val Phe Lys Val Asp Asn 370 375 380 Glu Asp Ile Tyr Glu Ser Arg His Glu Ile Lys Leu Pro Val Lys Trp 385 390 395 400 Thr Ala Pro Glu Ala Ile Arg Ser Asn Lys Phe Ser Ile Lys Ser Asp 405 410 415 Val Trp Ser Phe Gly Ile Leu Leu Tyr Glu Ile Ile Thr Tyr Gly Lys 420 425 430 Met Pro Tyr Ser Gly Met Thr Gly Ala Gln Val Ile Gln Met Leu Ala 435 440 445 Gln Asn Tyr Arg Leu Pro Gln Pro Ser Asn Cys Pro Gln Gln Phe Tyr 450 455 460 Asn Ile Met Leu Glu Cys Trp Asn Ala Glu Pro Lys Glu Arg Pro Thr 465 470 475 480 Phe Glu Thr Leu Arg Trp Lys Leu Glu Asp Tyr Phe Glu Thr Asp Ser 485 490 495 Ser Tyr Ser Asp Ala Asn Asn Phe Ile Arg 500 505 450 amino acids amino acid unknown unknown None not provided 7 Met Ser Ala Ile Gln Ala Ala Trp Pro Ser Gly Thr Glu Cys Ile Ala 1 5 10 15 Lys Tyr Asn Phe His Gly Thr Ala Glu Gln Asp Leu Pro Phe Cys Lys 20 25 30 Gly Asp Val Leu Thr Ile Val Ala Val Thr Lys Asp Pro Asn Trp Tyr 35 40 45 Lys Ala Lys Asn Lys Val Gly Arg Glu Gly Ile Ile Pro Ala Asn Tyr 50 55 60 Val Gln Lys Arg Glu Gly Val Lys Ala Gly Thr Lys Leu Ser Leu Met 65 70 75 80 Pro Trp Phe His Gly Lys Ile Thr Arg Glu Gln Ala Glu Arg Leu Leu 85 90 95 Tyr Pro Pro Glu Thr Gly Leu Phe Leu Val Arg Glu Ser Thr Asn Tyr 100 105 110 Pro Gly Asp Tyr Thr Leu Cys Val Ser Cys Asp Gly Lys Val Glu His 115 120 125 Tyr Arg Ile Met Tyr His Ala Ser Lys Leu Ser Ile Asp Glu Glu Val 130 135 140 Tyr Phe Glu Asn Leu Met Gln Leu Val Glu His Tyr Thr Ser Asp Ala 145 150 155 160 Asp Gly Leu Cys Thr Arg Leu Ile Lys Pro Lys Val Met Glu Gly Thr 165 170 175 Val Ala Ala Gln Asp Glu Phe Tyr Arg Ser Gly Trp Ala Leu Asn Met 180 185 190 Lys Glu Leu Lys Leu Leu Gln Thr Ile Gly Lys Gly Glu Phe Gly Asp 195 200 205 Val Met Leu Gly Asp Tyr Arg Gly Asn Lys Val Ala Val Lys Cys Ile 210 215 220 Lys Asn Asp Ala Thr Ala Gln Ala Phe Leu Ala Glu Ala Ser Val Met 225 230 235 240 Thr Gln Leu Arg His Ser Asn Leu Val Gln Leu Leu Gly Val Ile Val 245 250 255 Glu Glu Lys Gly Gly Leu Tyr Ile Val Thr Glu Tyr Met Ala Lys Gly 260 265 270 Ser Leu Val Asp Tyr Leu Arg Ser Arg Gly Arg Ser Val Leu Gly Gly 275 280 285 Asp Cys Leu Leu Lys Phe Ser Leu Asp Val Cys Glu Ala Met Glu Tyr 290 295 300 Leu Glu Gly Asn Asn Phe Val His Arg Asp Leu Ala Ala Arg Asn Val 305 310 315 320 Leu Val Ser Glu Asp Asn Val Ala Lys Val Ser Asp Phe Gly Leu Thr 325 330 335 Lys Glu Ala Ser Ser Thr Gln Asp Thr Gly Lys Leu Pro Val Lys Trp 340 345 350 Thr Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser Thr Lys Ser Asp 355 360 365 Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile Tyr Ser Phe Gly Arg 370 375 380 Val Pro Tyr Pro Arg Ile Pro Leu Lys Asp Val Val Pro Arg Val Glu 385 390 395 400 Lys Gly Tyr Lys Met Asp Ala Pro Asp Gly Cys Pro Pro Ala Val Tyr 405 410 415 Glu Val Met Lys Asn Cys Trp His Leu Asp Ala Ala Met Arg Pro Ser 420 425 430 Phe Leu Gln Leu Arg Glu Gln Leu Glu His Ile Lys Thr His Glu Leu 435 440 445 His Leu 450 659 amino acids amino acid unknown unknown None not provided 8 Met Ala Ala Val Ile Leu Glu Ser Ile Phe Leu Lys Arg Ser Gln Gln 1 5 10 15 Lys Lys Lys Thr Ser Pro Leu Asn Phe Lys Lys Arg Leu Phe Leu Leu 20 25 30 Thr Val His Lys Leu Ser Tyr Tyr Glu Tyr Asp Phe Glu Arg Gly Arg 35 40 45 Arg Gly Ser Lys Lys Gly Ser Ile Asp Val Glu Lys Ile Thr Cys Val 50 55 60 Glu Thr Val Val Pro Glu Lys Asn Pro Pro Pro Glu Arg Gln Ile Pro 65 70 75 80 Arg Arg Gly Glu Glu Ser Ser Glu Met Glu Gln Ile Ser Ile Ile Glu 85 90 95 Arg Phe Pro Tyr Pro Phe Gln Val Val Tyr Asp Glu Gly Pro Leu Tyr 100 105 110 Val Phe Ser Pro Thr Glu Glu Leu Arg Lys Arg Trp Ile His Gln Leu 115 120 125 Lys Asn Val Ile Arg Tyr Asn Ser Asp Leu Val Gln Lys Tyr His Pro 130 135 140 Cys Phe Trp Ile Asp Gly Gln Tyr Leu Cys Cys Ser Gln Thr Ala Lys 145 150 155 160 Asn Ala Met Gly Cys Gln Ile Leu Glu Asn Arg Asn Gly Ser Leu Lys 165 170 175 Pro Gly Ser Ser His Arg Lys Thr Lys Lys Pro Leu Pro Pro Thr Pro 180 185 190 Glu Glu Asp Gln Ile Leu Lys Lys Pro Leu Pro Pro Glu Pro Ala Ala 195 200 205 Ala Pro Val Ser Thr Ser Glu Leu Lys Lys Val Val Ala Leu Tyr Asp 210 215 220 Tyr Met Pro Met Asn Ala Asn Asp Leu Gln Leu Arg Lys Gly Asp Glu 225 230 235 240 Tyr Phe Ile Leu Glu Glu Ser Asn Leu Pro Trp Trp Arg Ala Arg Asp 245 250 255 Lys Asn Gly Gln Glu Gly Tyr Ile Pro Ser Asn Tyr Val Thr Glu Ala 260 265 270 Glu Asp Ser Ile Glu Met Tyr Glu Trp Tyr Ser Lys His Met Thr Arg 275 280 285 Ser Gln Ala Glu Gln Leu Leu Lys Gln Glu Gly Lys Glu Gly Gly Phe 290 295 300 Ile Val Arg Asp Ser Ser Lys Ala Gly Lys Tyr Thr Val Ser Val Phe 305 310 315 320 Ala Lys Ser Thr Gly Asp Pro Gln Gly Val Ile Arg His Tyr Val Val 325 330 335 Cys Ser Thr Pro Gln Ser Gln Tyr Tyr Leu Ala Glu Lys His Leu Phe 340 345 350 Ser Thr Ile Pro Glu Leu Ile Asn Tyr His Gln His Asn Ser Ala Gly 355 360 365 Leu Ile Ser Arg Leu Lys Tyr Pro Val Ser Gln Gln Asn Lys Asn Ala 370 375 380 Pro Ser Thr Ala Gly Leu Gly Tyr Gly Ser Trp Glu Ile Asp Pro Lys 385 390 395 400 Asp Leu Thr Phe Leu Lys Glu Leu Gly Thr Gly Gln Phe Gly Val Val 405 410 415 Lys Tyr Gly Lys Trp Arg Gly Gln Tyr Asp Val Ala Ile Lys Met Ile 420 425 430 Lys Glu Gly Ser Met Ser Glu Asp Glu Phe Ile Glu Glu Ala Lys Val 435 440 445 Met Met Asn Leu Ser His Glu Lys Leu Val Gln Leu Tyr Gly Val Cys 450 455 460 Thr Lys Gln Arg Pro Ile Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly 465 470 475 480 Cys Leu Leu Asn Tyr Leu Arg Glu Met Arg His Arg Phe Gln Thr Gln 485 490 495 Gln Leu Leu Glu Met Cys Lys Asp Val Cys Glu Ala Met Glu Tyr Leu 500 505 510 Glu Ser Lys Gln Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys Leu 515 520 525 Val Asn Asp Gln Gly Val Val Lys Val Ser Asp Phe Gly Leu Ser Arg 530 535 540 Tyr Val Leu Asp Asp Glu Tyr Thr Ser Ser Val Gly Ser Lys Phe Pro 545 550 555 560 Val Arg Trp Ser Pro Pro Glu Val Leu Met Tyr Ser Lys Phe Ser Ser 565 570 575 Lys Ser Asp Ile Trp Ala Phe Gly Val Leu Met Trp Glu Ile Tyr Ser 580 585 590 Leu Gly Lys Met Pro Tyr Glu Arg Phe Thr Asn Ser Glu Thr Ala Glu 595 600 605 His Ile Ala Gln Gly Leu Arg Leu Tyr Arg Pro His Leu Ala Ser Glu 610 615 620 Lys Val Tyr Thr Ile Met Tyr Ser Cys Trp His Glu Lys Ala Asp Glu 625 630 635 640 Arg Pro Thr Phe Lys Ile Leu Leu Ser Asn Ile Leu Asp Val Met Asp 645 650 655 Glu Glu Ser 620 amino acids amino acid unknown unknown not provided 9 Met Asn Asn Phe Ile Leu Leu Glu Glu Gln Leu Ile Lys Lys Ser Gln 1 5 10 15 Gln Lys Arg Arg Thr Ser Pro Ser Asn Phe Lys Val Arg Phe Phe Val 20 25 30 Leu Thr Lys Ala Ser Leu Ala Tyr Phe Glu Asp Arg His Gly Lys Lys 35 40 45 Arg Thr Leu Lys Gly Ser Ile Glu Leu Ser Arg Ile Lys Cys Val Glu 50 55 60 Ile Val Lys Ser Asp Ile Ser Ile Pro Cys His Tyr Lys Tyr Pro Phe 65 70 75 80 Gln Val Val His Asp Asn Tyr Leu Leu Tyr Val Phe Ala Pro Asp Arg 85 90 95 Glu Ser Arg Gln Arg Trp Val Leu Ala Leu Lys Glu Glu Thr Arg Asn 100 105 110 Asn Asn Ser Leu Val Pro Lys Tyr His Pro Asn Phe Trp Met Asp Gly 115 120 125 Lys Trp Arg Cys Cys Ser Gln Leu Glu Lys Leu Ala Thr Gly Cys Ala 130 135 140 Gln Tyr Asp Pro Thr Lys Asn Ala Ser Lys Lys Pro Leu Pro Pro Thr 145 150 155 160 Pro Glu Asp Asn Arg Arg Pro Leu Trp Glu Pro Glu Glu Thr Val Val 165 170 175 Ile Ala Leu Tyr Asp Tyr Gln Thr Asn Asp Pro Gln Glu Leu Ala Leu 180 185 190 Arg Arg Asn Glu Glu Tyr Cys Leu Leu Asp Ser Ser Glu Ile His Trp 195 200 205 Trp Arg Val Gln Asp Arg Asn Gly His Glu Gly Tyr Val Pro Ser Ser 210 215 220 Tyr Leu Val Glu Lys Ser Pro Asn Asn Leu Glu Thr Tyr Glu Trp Tyr 225 230 235 240 Asn Lys Ser Ile Ser Arg Asp Lys Ala Glu Lys Leu Leu Leu Asp Thr 245 250 255 Gly Lys Glu Gly Ala Phe Met Val Arg Asp Ser Arg Thr Ala Gly Thr 260 265 270 Tyr Thr Val Ser Val Phe Thr Lys Ala Val Val Ser Glu Asn Asn Pro 275 280 285 Cys Ile Lys His Tyr His Ile Lys Glu Thr Asn Asp Asn Pro Lys Arg 290 295 300 Tyr Tyr Val Ala Glu Lys Tyr Val Phe Asp Ser Ile Pro Leu Leu Ile 305 310 315 320 Asn Tyr His Gln His Asn Gly Gly Gly Leu Val Thr Arg Leu Arg Tyr 325 330 335 Pro Val Cys Phe Gly Arg Gln Lys Ala Pro Val Thr Ala Gly Leu Arg 340 345 350 Tyr Gly Lys Trp Val Ile Asp Pro Ser Glu Leu Thr Phe Val Gln Glu 355 360 365 Ile Gly Ser Gly Gln Phe Gly Leu Val His Leu Gly Tyr Trp Leu Asn 370 375 380 Lys Asp Lys Val Ala Ile Lys Thr Ile Arg Glu Gly Ala Met Ser Glu 385 390 395 400 Glu Asp Phe Ile Glu Glu Ala Glu Val Met Met Lys Leu Ser His Pro 405 410 415 Lys Leu Val Gln Leu Tyr Gly Val Cys Leu Glu Gln Ala Pro Ile Cys 420 425 430 Leu Val Phe Glu Phe Met Glu His Gly Cys Leu Ser Asp Tyr Leu Arg 435 440 445 Thr Gln Arg Gly Leu Phe Ala Ala Glu Thr Leu Leu Gly Met Cys Leu 450 455 460 Asp Val Cys Glu Gly Met Ala Tyr Leu Glu Glu Ala Cys Val Ile His 465 470 475 480 Arg Asp Leu Ala Ala Arg Asn Cys Leu Val Gly Glu Asn Gln Val Ile 485 490 495 Lys Val Ser Asp Phe Gly Met Thr Arg Phe Val Leu Asp Asp Gln Tyr 500 505 510 Thr Ser Ser Thr Gly Thr Lys Phe Pro Val Lys Trp Ala Ser Pro Glu 515 520 525 Val Phe Ser Phe Ser Arg Tyr Ser Ser Lys Ser Asp Val Trp Ser Phe 530 535 540 Gly Val Leu Met Trp Glu Val Phe Ser Glu Gly Lys Ile Pro Tyr Glu 545 550 555 560 Asn Arg Ser Asn Ser Glu Val Val Glu Asp Ile Ser Thr Gly Phe Arg 565 570 575 Leu Tyr Lys Pro Arg Leu Ala Ser Thr His Val Tyr Gln Ile Met Asn 580 585 590 His Cys Trp Lys Glu Arg Pro Glu Asp Arg Pro Ala Phe Ser Arg Leu 595 600 605 Leu Arg Gln Leu Ala Glu Ile Ala Glu Ser Gly Leu 610 615 620 527 amino acids amino acid unknown unknown None not provided 10 Met Met Val Ser Phe Pro Val Lys Ile Asn Phe His Ser Ser Pro Gln 1 5 10 15 Ser Arg Asp Arg Trp Val Lys Lys Leu Lys Glu Glu Ile Lys Asn Asn 20 25 30 Asn Asn Ile Met Ile Lys Tyr His Pro Lys Phe Trp Ala Asp Gly Ser 35 40 45 Tyr Gln Cys Cys Arg Gln Thr Glu Lys Leu Ala Pro Gly Cys Glu Lys 50 55 60 Tyr Asn Leu Phe Glu Ser Ser Ile Arg Lys Thr Leu Pro Pro Ala Pro 65 70 75 80 Glu Ile Lys Lys Arg Arg Pro Pro Pro Pro Ile Pro Pro Glu Glu Glu 85 90 95 Asn Thr Glu Glu Ile Val Val Ala Met Tyr Asp Phe Gln Ala Thr Glu 100 105 110 Ala His Asp Leu Arg Leu Glu Arg Gly Gln Glu Tyr Ile Ile Leu Glu 115 120 125 Lys Asn Asp Leu His Trp Trp Arg Ala Arg Asp Lys Tyr Gly Trp Tyr 130 135 140 Cys Arg Asn Thr Asn Arg Ser Lys Ala Glu Gln Leu Leu Arg Thr Glu 145 150 155 160 Asp Lys Glu Gly Gly Phe Met Val Arg Asp Ser Ser Gln Pro Gly Leu 165 170 175 Tyr Thr Val Ser Leu Tyr Thr Lys Phe Gly Gly Glu Gly Ser Ser Gly 180 185 190 Phe Arg His Tyr His Ile Lys Glu Thr Ala Thr Ser Pro Lys Lys Tyr 195 200 205 Tyr Leu Ala Glu Lys His Ala Phe Gly Ser Ile Pro Glu Ile Ile Glu 210 215 220 Tyr His Lys His Asn Ala Ala Gly Leu Val Thr Arg Leu Arg Tyr Pro 225 230 235 240 Val Ser Thr Lys Gly Lys Asn Ala Pro Thr Thr Ala Gly Phe Ser Tyr 245 250 255 Asp Lys Trp Glu Ile Asn Pro Ser Glu Leu Thr Phe Met Arg Glu Leu 260 265 270 Gly Ser Gly Leu Phe Gly Val Val Arg Leu Gly Lys Trp Arg Ala Gln 275 280 285 Tyr Lys Val Ala Ile Lys Ala Ile Arg Glu Gly Ala Met Cys Glu Glu 290 295 300 Asp Phe Ile Glu Glu Ala Lys Val Met Met Lys Leu Thr His Pro Lys 305 310 315 320 Leu Val Gln Leu Tyr Gly Val Cys Thr Gln Gln Lys Pro Ile Tyr Ile 325 330 335 Val Thr Glu Phe Met Glu Arg Gly Cys Leu Leu Asn Phe Leu Arg Gln 340 345 350 Arg Gln Gly His Phe Ser Arg Asp Met Leu Leu Ser Met Cys Gln Asp 355 360 365 Val Cys Glu Gly Met Glu Tyr Leu Glu Arg Asn Ser Phe Ile His Arg 370 375 380 Asp Leu Ala Ala Arg Asn Cys Leu Val Asn Glu Ala Gly Val Val Lys 385 390 395 400 Val Ser Asp Phe Gly Met Ala Arg Tyr Val Leu Asp Asp Gln Tyr Thr 405 410 415 Ser Ser Ser Gly Ala Lys Phe Pro Val Lys Trp Cys Pro Pro Glu Val 420 425 430 Phe Asn Tyr Ser Arg Phe Ser Ser Lys Ser Asp Val Trp Ser Phe Gly 435 440 445 Val Leu Met Trp Glu Ile Phe Thr Glu Gly Arg Met Pro Phe Glu Lys 450 455 460 Asn Thr Asn Tyr Glu Val Val Thr Met Val Thr Arg Gly His Arg Leu 465 470 475 480 His Arg Pro Lys Leu Ala Thr Lys Tyr Leu Tyr Glu Val Met Leu Arg 485 490 495 Cys Trp Gln Glu Arg Pro Glu Gly Arg Pro Ser Phe Glu Asp Leu Leu 500 505 510 Arg Thr Ile Asp Glu Leu Val Glu Cys Glu Glu Thr Phe Gly Arg 515 520 525 537 amino acids amino acid unknown unknown None not provided 11 Met Gly Cys Val Gln Cys Lys Asp Lys Glu Ala Thr Lys Leu Thr Glu 1 5 10 15 Glu Arg Asp Gly Ser Leu Asn Gln Ser Ser Gly Tyr Arg Tyr Gly Thr 20 25 30 Asp Pro Thr Pro Gln His Tyr Pro Ser Phe Gly Val Thr Ser Ile Pro 35 40 45 Asn Tyr Asn Asn Phe His Ala Ala Gly Gly Gln Gly Leu Thr Val Phe 50 55 60 Gly Gly Val Asn Ser Ser Ser His Thr Gly Thr Leu Arg Thr Arg Gly 65 70 75 80 Gly Thr Gly Val Thr Leu Phe Val Ala Leu Tyr Asp Tyr Glu Ala Arg 85 90 95 Thr Glu Asp Asp Leu Ser Phe His Lys Gly Glu Lys Phe Gln Ile Leu 100 105 110 Asn Ser Ser Glu Gly Asp Trp Trp Glu Ala Arg Ser Leu Thr Thr Gly 115 120 125 Glu Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Val Asp Ser Ile 130 135 140 Gln Ala Glu Glu Trp Tyr Phe Gly Lys Leu Gly Arg Lys Asp Ala Glu 145 150 155 160 Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg Gly Thr Phe Leu Ile Arg 165 170 175 Glu Ser Glu Thr Thr Lys Gly Ala Tyr Ser Leu Ser Ile Arg Asp Trp 180 185 190 Asp Asp Met Lys Gly Asp His Val Lys His Tyr Lys Ile Arg Lys Leu 195 200 205 Asp Asn Gly Gly Tyr Tyr Ile Thr Thr Arg Ala Gln Phe Glu Thr Leu 210 215 220 Gln Gln Leu Val Gln His Tyr Ser Glu Arg Ala Ala Gly Leu Cys Cys 225 230 235 240 Arg Leu Val Val Pro Cys His Lys Gly Met Pro Arg Leu Thr Asp Leu 245 250 255 Ser Val Lys Thr Lys Asp Val Trp Glu Ile Pro Arg Glu Ser Leu Gln 260 265 270 Leu Ile Lys Arg Leu Gly Asn Gly Gln Phe Gly Glu Val Trp Met Gly 275 280 285 Thr Trp Asn Gly Asn Thr Lys Val Ala Ile Lys Thr Leu Lys Pro Gly 290 295 300 Thr Met Ser Pro Glu Ser Phe Leu Glu Glu Ala Gln Ile Met Lys Lys 305 310 315 320 Leu Lys His Asp Lys Leu Val Gln Leu Tyr Ala Val Val Ser Glu Glu 325 330 335 Pro Ile Tyr Ile Val Thr Glu Tyr Met Asn Lys Gly Ser Leu Leu Asp 340 345 350 Phe Leu Lys Asp Gly Glu Gly Arg Ala Leu Lys Leu Pro Asn Leu Val 355 360 365 Asp Met Ala Ala Gln Val Ala Ala Gly Met Ala Tyr Ile Glu Arg Met 370 375 380 Asn Tyr Ile His Arg Asp Leu Arg Ser Ala Asn Ile Leu Val Gly Asn 385 390 395 400 Gly Leu Ile Cys Lys Ile Ala Asp Phe Gly Leu Ala Arg Leu Ile Glu 405 410 415 Asp Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Phe Pro Ile Lys Trp 420 425 430 Thr Ala Pro Glu Ala Ala Leu Tyr Gly Arg Phe Thr Ile Lys Ser Asp 435 440 445 Val Trp Ser Phe Gly Ile Leu Leu Thr Glu Leu Val Thr Lys Gly Arg 450 455 460 Val Pro Tyr Pro Gly Met Asn Asn Arg Glu Val Leu Glu Gln Val Glu 465 470 475 480 Arg Gly Tyr Arg Met Pro Cys Pro Gln Asp Cys Pro Ile Ser Leu His 485 490 495 Glu Leu Met Ile His Cys Trp Lys Lys Asp Pro Glu Glu Arg Pro Thr 500 505 510 Phe Glu Tyr Leu Gln Ser Phe Leu Glu Asp Tyr Phe Thr Ala Thr Glu 515 520 525 Pro Gln Tyr Gln Pro Gly Glu Asn Leu 530 535 536 amino acids amino acid unknown unknown None not provided 12 Met Gly Cys Val His Cys Lys Glu Lys Ile Ser Gly Lys Gly Gln Gly 1 5 10 15 Gly Ser Gly Thr Gly Thr Pro Ala His Pro Pro Ser Gln Tyr Asp Pro 20 25 30 Asp Pro Thr Gln Leu Ser Gly Ala Phe Thr His Ile Pro Asp Phe Asn 35 40 45 Asn Phe His Ala Ala Ala Val Ser Pro Pro Val Pro Phe Ser Gly Pro 50 55 60 Gly Phe Tyr Pro Cys Asn Thr Leu Gln Ala His Ser Ser Ile Thr Gly 65 70 75 80 Gly Gly Val Thr Leu Phe Ile Ala Leu Tyr Asp Tyr Glu Ala Arg Thr 85 90 95 Glu Asp Asp Leu Ser Phe Gln Lys Gly Glu Lys Phe His Ile Ile Asn 100 105 110 Asn Thr Glu Gly Asp Trp Trp Glu Ala Arg Ser Leu Ser Ser Gly Ala 115 120 125 Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Val Asp Ser Ile Gln 130 135 140 Ala Glu Glu Trp Tyr Phe Gly Lys Ile Gly Arg Lys Asp Ala Glu Arg 145 150 155 160 Gln Leu Leu Cys His Gly Asn Cys Arg Gly Thr Phe Leu Ile Arg Glu 165 170 175 Ser Glu Thr Thr Lys Gly Ala Tyr Ser Leu Ser Ile Arg Asp Trp Asp 180 185 190 Glu Ala Lys Gly Asp His Val Lys His Tyr Lys Ile Arg Lys Leu Asp 195 200 205 Ser Gly Gly Tyr Tyr Ile Thr Thr Arg Ala Gln Phe Asp Thr Ile Gln 210 215 220 Gln Leu Val Gln His Tyr Ile Glu Arg Ala Ala Gly Leu Cys Cys Arg 225 230 235 240 Leu Ala Val Pro Cys Pro Lys Gly Thr Pro Lys Leu Ala Asp Leu Ser 245 250 255 Val Lys Thr Lys Asp Val Trp Glu Ile Pro Arg Glu Ser Leu Gln Leu 260 265 270 Leu Gln Lys Leu Gly Asn Gly Gln Phe Gly Glu Val Trp Met Gly Thr 275 280 285 Trp Asn Gly Thr Thr Lys Val Ala Val Lys Thr Leu Lys Pro Gly Thr 290 295 300 Met Ser Pro Glu Ala Phe Leu Glu Glu Ala Gln Ile Met Lys Arg Leu 305 310 315 320 Arg His Asp Lys Leu Val Gln Leu Tyr Ala Val Val Ser Glu Glu Pro 325 330 335 Ile Tyr Ile Val Thr Glu Phe Met Ser Gln Gly Ser Leu Leu Asp Phe 340 345 350 Leu Lys Asp Gly Asp Gly Arg Tyr Leu Lys Leu Pro Gln Leu Val Asp 355 360 365 Met Ala Ala Gln Ile Ala Ala Gly Met Ala Tyr Ile Glu Arg Met Asn 370 375 380 Tyr Ile His Arg Asp Leu Arg Ala Ala Asn Ile Leu Val Gly Asp Asn 385 390 395 400 Leu Val Cys Lys Ile Ala Asp Phe Gly Leu Ala Arg Leu Ile Glu Asp 405 410 415 Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Phe Pro Ile Lys Trp Thr 420 425 430 Ala Pro Glu Ala Ala Leu Phe Gly Lys Phe Thr Ile Lys Ser Asp Val 435 440 445 Trp Ser Phe Gly Ile Leu Leu Thr Glu Leu Val Thr Lys Gly Arg Val 450 455 460 Pro Tyr Pro Gly Met Asn Asn Arg Glu Val Leu Glu Gln Val Glu Arg 465 470 475 480 Gly Tyr Arg Met Gln Cys Pro Gly Gly Cys Pro Pro Ser Leu His Asp 485 490 495 Val Met Val Gln Cys Trp Lys Arg Glu Pro Glu Glu Arg Pro Thr Phe 500 505 510 Glu Tyr Leu Gln Ser Phe Leu Glu Asp Tyr Phe Thr Ala Thr Glu Pro 515 520 525 Gln Tyr Gln Pro Gly Asp Asn Gln 530 535 536 amino acids amino acid unknown unknown None not provided 13 Met Gly Ser Asn Lys Ser Lys Pro Lys Asp Ala Ser Gln Arg Arg Arg 1 5 10 15 Ser Leu Glu Pro Ala Glu Asn Val His Gly Ala Gly Gly Gly Ala Phe 20 25 30 Pro Ala Ser Gln Thr Pro Ser Lys Pro Ala Ser Ala Asp Gly His Arg 35 40 45 Gly Pro Ser Ala Ala Phe Ala Pro Ala Ala Ala Glu Pro Lys Leu Phe 50 55 60 Gly Gly Phe Asn Ser Ser Asp Thr Val Thr Ser Pro Gln Arg Ala Gly 65 70 75 80 Pro Leu Ala Gly Gly Val Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu 85 90 95 Ser Arg Thr Glu Thr Asp Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln 100 105 110 Ile Val Asn Asn Thr Glu Gly Asp Trp Trp Leu Ala His Ser Leu Ser 115 120 125 Thr Gly Gln Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Ser Asp 130 135 140 Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys Ile Thr Arg Arg Glu 145 150 155 160 Ser Glu Arg Leu Leu Leu Asn Ala Glu Asn Pro Arg Gly Thr Phe Leu 165 170 175 Val Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Cys Leu Ser Val Ser 180 185 190 Asp Phe Asp Asn Ala Lys Gly Leu Asn Val Lys His Tyr Lys Ile Arg 195 200 205 Lys Leu Asp Ser Gly Gly Phe Tyr Ile Thr Ser Arg Thr Gln Phe Asn 210 215 220 Ser Leu Gln Gln Leu Val Ala Tyr Tyr Ser Lys His Ala Asp Gly Leu 225 230 235 240 Cys His Arg Leu Thr Thr Val Cys Pro Thr Ser Lys Pro Gln Thr Gln 245 250 255 Gly Leu Ala Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser Leu Arg Leu 260 265 270 Glu Val Lys Leu Gly Gln Gly Cys Phe Gly Glu Val Trp Met Gly Thr 275 280 285 Trp Asn Gly Thr Thr Arg Val Ala Ile Lys Thr Leu Lys Pro Gly Thr 290 295 300 Met Ser Pro Glu Ala Phe Leu Gln Glu Ala Gln Val Met Lys Lys Leu 305 310 315 320 Arg His Glu Lys Leu Val Gln Leu Tyr Ala Val Val Ser Glu Glu Pro 325 330 335 Ile Tyr Ile Val Thr Glu Tyr Met Ser Lys Gly Ser Leu Leu Asp Phe 340 345 350 Leu Lys Gly Glu Thr Gly Lys Tyr Leu Arg Leu Pro Gln Leu Val Asp 355 360 365 Met Ala Ala Gln Ile Ala Ser Gly Met Ala Tyr Val Glu Arg Met Asn 370 375 380 Tyr Val His Arg Asp Leu Arg Ala Ala Asn Ile Leu Val Gly Glu Asn 385 390 395 400 Leu Val Cys Lys Val Ala Asp Phe Gly Leu Ala Arg Leu Ile Glu Asp 405 410 415 Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Phe Pro Ile Lys Trp Thr 420 425 430 Ala Pro Glu Ala Ala Leu Tyr Gly Arg Phe Thr Ile Lys Ser Asp Val 435 440 445 Trp Ser Phe Gly Ile Leu Leu Thr Glu Leu Thr Thr Lys Gly Arg Val 450 455 460 Pro Tyr Pro Gly Met Val Asn Arg Glu Val Leu Asp Gln Val Glu Arg 465 470 475 480 Gly Tyr Arg Met Pro Cys Pro Pro Glu Cys Pro Glu Ser Leu His Asp 485 490 495 Leu Met Cys Gln Cys Trp Arg Lys Glu Pro Glu Glu Arg Pro Thr Phe 500 505 510 Glu Tyr Leu Gln Ala Phe Leu Glu Asp Tyr Phe Thr Ser Thr Glu Pro 515 520 525 Gln Tyr Gln Pro Gly Glu Asn Leu 530 535 543 amino acids amino acid unknown unknown None not provided 14 Met Gly Cys Ile Lys Ser Lys Glu Asn Lys Ser Pro Ala Ile Lys Tyr 1 5 10 15 Arg Pro Glu Asn Thr Pro Glu Pro Val Ser Thr Ser Val Ser His Tyr 20 25 30 Gly Ala Glu Pro Thr Thr Val Ser Pro Cys Pro Ser Ser Ser Ala Lys 35 40 45 Gly Thr Ala Val Asn Phe Ser Ser Leu Ser Met Thr Pro Phe Gly Gly 50 55 60 Ser Ser Gly Val Thr Pro Phe Gly Gly Ala Ser Ser Ser Phe Ser Val 65 70 75 80 Val Pro Ser Ser Tyr Pro Ala Gly Leu Thr Gly Gly Val Thr Ile Phe 85 90 95 Val Ala Leu Tyr Asp Tyr Glu Ala Arg Thr Thr Glu Asp Leu Ser Phe 100 105 110 Lys Lys Gly Glu Arg Phe Gln Ile Ile Asn Asn Thr Glu Gly Asp Trp 115 120 125 Trp Glu Ala Arg Ser Ile Ala Thr Gly Lys Asn Gly Tyr Ile Pro Ser 130 135 140 Asn Tyr Val Ala Pro Ala Asp Ser Ile Gln Ala Glu Glu Trp Tyr Phe 145 150 155 160 Gly Lys Met Gly Arg Lys Asp Ala Glu Arg Leu Leu Leu Asn Pro Gly 165 170 175 Asn Gln Arg Gly Ile Phe Leu Val Arg Glu Ser Glu Thr Thr Lys Gly 180 185 190 Ala Tyr Ser Leu Ser Ile Arg Asp Trp Asp Glu Ile Arg Gly Asp Asn 195 200 205 Val Lys His Tyr Lys Ile Arg Lys Leu Asp Asn Gly Gly Tyr Tyr Ile 210 215 220 Thr Thr Arg Ala Gln Phe Asp Thr Leu Gln Lys Leu Val Lys His Tyr 225 230 235 240 Thr Glu His Ala Asp Gly Leu Cys His Lys Leu Thr Thr Val Cys Pro 245 250 255 Thr Val Lys Pro Gln Thr Gln Gly Leu Ala Lys Asp Ala Trp Glu Ile 260 265 270 Pro Arg Glu Ser Leu Arg Leu Glu Val Lys Leu Gly Gln Gly Cys Phe 275 280 285 Gly Glu Val Trp Met Gly Thr Trp Asn Gly Thr Thr Lys Val Ala Ile 290 295 300 Lys Thr Leu Lys Pro Gly Thr Met Met Pro Glu Ala Phe Leu Gln Glu 305 310 315 320 Ala Gln Ile Met Lys Lys Leu Arg His Asp Lys Leu Val Pro Leu Tyr 325 330 335 Ala Val Val Ser Glu Glu Pro Ile Tyr Ile Val Thr Glu Phe Met Ser 340 345 350 Lys Gly Ser Leu Leu Asp Phe Leu Lys Glu Gly Asp Gly Lys Tyr Leu 355 360 365 Lys Leu Pro Gln Leu Val Asp Met Ala Ala Gln Ile Ala Asp Gly Met 370 375 380 Ala Tyr Ile Glu Arg Met Asn Tyr Ile His Arg Asp Leu Arg Ala Ala 385 390 395 400 Asn Ile Leu Val Gly Glu Asn Leu Val Cys Lys Ile Ala Asp Phe Gly 405 410 415 Leu Ala Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg Gln Gly Ala 420 425 430 Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ala Leu Tyr Gly Arg 435 440 445 Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Gln Thr Glu 450 455 460 Leu Val Thr Lys Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu 465 470 475 480 Val Leu Glu Gln Val Glu Arg Gly Tyr Arg Met Pro Cys Pro Gln Gly 485 490 495 Cys Pro Glu Ser Leu His Glu Leu Met Asn Leu Cys Trp Lys Lys Asp 500 505 510 Pro Asp Glu Arg Pro Thr Phe Glu Tyr Ile Gln Ser Phe Leu Glu Asp 515 520 525 Tyr Phe Thr Ala Thr Glu Pro Gln Tyr Gln Pro Gly Glu Asn Leu 530 535 540 529 amino acids amino acid unknown unknown None not provided 15 Met Gly Cys Val Phe Cys Lys Lys Leu Glu Pro Val Ala Thr Ala Lys 1 5 10 15 Glu Asp Ala Gly Leu Glu Gly Asp Phe Arg Ser Tyr Gly Ala Ala Asp 20 25 30 His Tyr Gly Pro Asp Pro Thr Lys Ala Arg Pro Ala Ser Ser Phe Ala 35 40 45 His Ile Pro Asn Tyr Ser Asn Phe Ser Ser Gln Ala Ile Asn Pro Gly 50 55 60 Phe Leu Asp Ser Gly Thr Ile Arg Gly Val Ser Gly Ile Gly Val Thr 65 70 75 80 Leu Phe Ile Ala Leu Tyr Asp Tyr Glu Ala Arg Thr Glu Asp Asp Leu 85 90 95 Thr Phe Thr Lys Gly Glu Lys Phe His Ile Leu Asn Asn Thr Glu Gly 100 105 110 Asp Trp Trp Glu Ala Arg Ser Leu Ser Ser Gly Lys Thr Gly Cys Ile 115 120 125 Pro Ser Asn Tyr Val Ala Pro Val Asp Ser Ile Gln Ala Glu Glu Trp 130 135 140 Tyr Phe Gly Lys Ile Gly Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser 145 150 155 160 Pro Gly Asn Pro Gln Gly Ala Phe Leu Ile Arg Glu Ser Glu Thr Thr 165 170 175 Lys Gly Ala Tyr Ser Leu Ser Ile Arg Asp Trp Asp Gln Thr Arg Gly 180 185 190 Asp His Val Lys His Tyr Lys Ile Arg Lys Leu Asp Met Gly Gly Tyr 195 200 205 Tyr Ile Thr Thr Arg Val Gln Phe Asn Ser Val Gln Glu Leu Val Gln 210 215 220 His Tyr Met Glu Val Asn Asp Gly Leu Cys Asn Leu Leu Ile Ala Pro 225 230 235 240 Cys Thr Ile Met Lys Pro Gln Thr Leu Gly Leu Ala Lys Asp Ala Trp 245 250 255 Glu Ile Ser Arg Ser Ser Ile Thr Leu Glu Arg Arg Leu Gly Thr Gly 260 265 270 Cys Phe Gly Asp Val Trp Leu Gly Thr Trp Asn Gly Ser Thr Lys Val 275 280 285 Ala Val Lys Thr Leu Lys Pro Gly Thr Met Ser Pro Lys Ala Phe Leu 290 295 300 Glu Glu Ala Gln Val Met Lys Leu Leu Arg His Asp Lys Leu Val Gln 305 310 315 320 Leu Tyr Ala Val Val Ser Glu Glu Pro Ile Tyr Ile Val Thr Glu Phe 325 330 335 Met Cys His Gly Ser Leu Leu Asp Phe Leu Lys Asn Pro Glu Gly Gln 340 345 350 Asp Leu Arg Leu Pro Gln Leu Val Asp Met Ala Ala Gln Val Ala Glu 355 360 365 Gly Met Ala Tyr Met Glu Arg Met Asn Tyr Ile His Arg Asp Leu Arg 370 375 380 Ala Ala Asn Ile Leu Val Gly Glu Arg Leu Ala Cys Lys Ile Ala Asp 385 390 395 400 Phe Gly Leu Ala Arg Leu Ile Lys Asp Asp Glu Tyr Asn Pro Cys Gln 405 410 415 Gly Ser Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ala Leu Phe 420 425 430 Gly Arg Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Leu 435 440 445 Thr Glu Leu Ile Thr Lys Gly Arg Ile Pro Tyr Pro Gly Met Asn Lys 450 455 460 Arg Glu Val Leu Glu Gln Val Glu Gln Gly Tyr His Met Pro Cys Pro 465 470 475 480 Pro Gly Cys Pro Ala Ser Leu Tyr Glu Ala Met Glu Gln Thr Trp Arg 485 490 495 Leu Asp Pro Glu Glu Arg Pro Thr Phe Glu Tyr Leu Gln Ser Phe Leu 500 505 510 Glu Asp Tyr Phe Thr Ser Ala Glu Pro Gln Tyr Gln Pro Gly Asp Gln 515 520 525 Thr 512 amino acids amino acid unknown unknown None not provided 16 Met Gly Cys Ile Lys Ser Lys Gly Lys Asp Ser Leu Ser Asp Asp Gly 1 5 10 15 Val Asp Leu Lys Thr Gln Pro Val Arg Asn Thr Glu Arg Thr Ile Tyr 20 25 30 Val Arg Asp Pro Thr Ser Asn Lys Gln Gln Arg Pro Val Pro Glu Ser 35 40 45 Gln Leu Leu Pro Gly Gln Arg Phe Gln Thr Lys Asp Pro Glu Glu Gln 50 55 60 Gly Asp Ile Val Val Ala Leu Tyr Pro Tyr Asp Gly Ile His Pro Asp 65 70 75 80 Asp Leu Ser Phe Lys Lys Gly Glu Lys Met Lys Val Leu Glu Glu His 85 90 95 Gly Glu Trp Trp Lys Ala Lys Ser Leu Leu Thr Lys Lys Glu Gly Phe 100 105 110 Ile Pro Ser Asn Tyr Val Ala Lys Leu Asn Thr Leu Glu Thr Glu Glu 115 120 125 Trp Phe Phe Lys Asp Ile Thr Arg Lys Asp Ala Glu Arg Gln Leu Leu 130 135 140 Ala Pro Gly Asn Ser Ala Gly Ala Phe Leu Ile Arg Glu Ser Glu Thr 145 150 155 160 Leu Lys Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Pro Val His 165 170 175 Gly Asp Val Ile Lys His Tyr Lys Ile Arg Ser Leu Asp Asn Gly Gly 180 185 190 Tyr Tyr Ile Ser Pro Arg Ile Thr Phe Pro Cys Ile Ser Asp Met Ile 195 200 205 Lys His Tyr Gln Lys Gln Ala Asp Gly Leu Cys Arg Arg Leu Glu Lys 210 215 220 Ala Cys Ile Ser Pro Lys Pro Gln Lys Pro Trp Asp Lys Asp Ala Trp 225 230 235 240 Glu Ile Pro Arg Glu Ser Ile Lys Leu Val Lys Arg Leu Gly Ala Gly 245 250 255 Gln Phe Gly Glu Val Trp Met Gly Tyr Tyr Asn Asn Ser Thr Lys Val 260 265 270 Ala Val Lys Thr Leu Lys Pro Gly Thr Met Ser Val Gln Ala Phe Leu 275 280 285 Glu Glu Ala Asn Leu Met Lys Thr Leu Gln His Asp Lys Leu Val Arg 290 295 300 Leu Tyr Ala Val Val Thr Arg Glu Glu Pro Ile Tyr Ile Ile Thr Glu 305 310 315 320 Tyr Met Ala Lys Gly Ser Leu Leu Asp Phe Leu Lys Ser Asp Glu Gly 325 330 335 Gly Lys Val Leu Leu Pro Lys Leu Ile Asp Phe Ser Ala Gln Ile Ala 340 345 350 Glu Gly Met Ala Tyr Ile Glu Arg Lys Asn Tyr Ile His Arg Asp Leu 355 360 365 Arg Ala Ala Asn Val Leu Val Ser Glu Ser Leu Met Cys Lys Ile Ala 370 375 380 Asp Phe Gly Leu Ala Arg Val Ile Glu Asp Asn Glu Tyr Thr Ala Arg 385 390 395 400 Glu Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ile Asn 405 410 415 Phe Gly Cys Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu 420 425 430 Leu Tyr Glu Ile Val Thr Tyr Gly Lys Ile Pro Tyr Pro Gly Arg Thr 435 440 445 Asn Ala Asp Val Met Thr Ala Leu Ser Gln Gly Tyr Arg Met Pro Arg 450 455 460 Val Glu Asn Cys Pro Asp Glu Leu Tyr Asp Ile Met Lys Met Cys Trp 465 470 475 480 Lys Glu Lys Ala Glu Glu Arg Pro Thr Phe Asp Tyr Leu Gln Ser Val 485 490 495 Leu Asp Asp Phe Tyr Thr Ala Thr Glu Gly Gln Tyr Gln Gln Gln Pro 500 505 510 505 amino acids amino acid unknown unknown not provided 17 Met Gly Ser Met Lys Ser Lys Phe Leu Gln Val Gly Gly Asn Thr Phe 1 5 10 15 Ser Lys Thr Glu Thr Ser Ala Ser Pro His Cys Pro Val Tyr Val Pro 20 25 30 Asp Pro Thr Ser Thr Ile Lys Pro Gly Pro Asn Ser His Asn Ser Asn 35 40 45 Thr Pro Gly Ile Arg Glu Ala Gly Ser Glu Asp Ile Ile Val Val Ala 50 55 60 Leu Tyr Asp Tyr Glu Ala Ile His His Glu Asp Leu Ser Phe Gln Lys 65 70 75 80 Gly Asp Gln Met Val Val Leu Glu Glu Ser Gly Glu Trp Trp Lys Ala 85 90 95 Arg Ser Leu Ala Thr Arg Lys Glu Gly Tyr Ile Pro Ser Asn Tyr Val 100 105 110 Ala Arg Val Asp Ser Leu Glu Thr Glu Glu Trp Phe Phe Lys Gly Ile 115 120 125 Ser Arg Lys Asp Ala Glu Arg Gln Leu Leu Ala Pro Gly Asn Met Leu 130 135 140 Gly Ser Phe Met Ile Arg Asp Ser Glu Thr Thr Lys Gly Ser Tyr Ser 145 150 155 160 Leu Ser Val Arg Asp Tyr Asp Pro Arg Gln Gly Asp Thr Val Lys His 165 170 175 Tyr Lys Ile Arg Thr Leu Asp Asn Gly Gly Phe Tyr Ile Ser Pro Arg 180 185 190 Ser Thr Phe Ser Thr Leu Gln Glu Leu Val Asp His Tyr Lys Lys Gly 195 200 205 Asn Asp Gly Leu Cys Gln Lys Leu Ser Val Pro Cys Met Ser Ser Lys 210 215 220 Pro Gln Lys Pro Trp Glu Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser 225 230 235 240 Leu Lys Leu Glu Lys Lys Leu Gly Ala Gly Gln Phe Gly Glu Val Trp 245 250 255 Met Ala Thr Tyr Asn Lys His Thr Lys Val Ala Val Lys Thr Met Lys 260 265 270 Pro Gly Ser Met Ser Val Glu Ala Phe Leu Ala Glu Ala Asn Val Met 275 280 285 Lys Thr Leu Gln His Asp Lys Leu Val Lys Leu His Ala Val Val Thr 290 295 300 Lys Glu Pro Ile Tyr Ile Ile Thr Glu Phe Met Ala Lys Gly Ser Leu 305 310 315 320 Leu Asp Phe Leu Lys Ser Asp Glu Gly Ser Lys Gln Pro Leu Pro Lys 325 330 335 Leu Ile Asp Phe Ser Ala Gln Ile Ala Glu Gly Met Ala Phe Ile Glu 340 345 350 Gln Arg Asn Tyr Ile His Arg Asp Leu Arg Ala Ala Asn Ile Leu Val 355 360 365 Ser Ala Ser Leu Val Cys Lys Ile Ala Asp Phe Gly Leu Ala Arg Val 370 375 380 Ile Glu Asp Asn Glu Tyr Thr Ala Arg Glu Gly Ala Lys Phe Pro Ile 385 390 395 400 Lys Trp Thr Ala Pro Glu Ala Ile Asn Phe Gly Ser Phe Thr Ile Lys 405 410 415 Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Met Glu Ile Val Thr Tyr 420 425 430 Gly Arg Ile Pro Tyr Pro Gly Met Ser Asn Pro Glu Val Ile Arg Ala 435 440 445 Leu Glu Arg Gly Tyr Arg Met Pro Arg Pro Glu Asn Cys Pro Glu Glu 450 455 460 Leu Tyr Asn Ile Met Met Arg Cys Trp Lys Asn Arg Pro Glu Glu Arg 465 470 475 480 Pro Thr Phe Glu Tyr Ile Gln Ser Val Leu Asp Asp Phe Tyr Thr Ala 485 490 495 Thr Glu Ser Gln Tyr Gln Gln Gln Pro 500 505 509 amino acids amino acid unknown unknown not provided 18 Met Gly Cys Gly Cys Ser Ser His Pro Glu Asp Asp Trp Met Glu Asn 1 5 10 15 Ile Asp Val Cys Glu Asn Cys His Tyr Pro Ile Val Pro Leu Asp Gly 20 25 30 Lys Gly Thr Leu Leu Ile Arg Asn Gly Ser Glu Val Arg Asp Pro Leu 35 40 45 Val Thr Tyr Glu Gly Ser Asn Pro Pro Ala Ser Pro Leu Gln Asp Asn 50 55 60 Leu Val Ile Ala Leu His Ser Tyr Glu Pro Ser His Asp Gly Asp Leu 65 70 75 80 Gly Phe Glu Lys Gly Glu Gln Leu Arg Ile Leu Glu Gln Ser Gly Glu 85 90 95 Trp Trp Lys Ala Gln Ser Leu Thr Thr Gly Gln Glu Gly Phe Ile Pro 100 105 110 Phe Asn Phe Val Ala Lys Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe 115 120 125 Phe Lys Asn Leu Ser Arg Lys Asp Ala Glu Arg Gln Leu Leu Ala Pro 130 135 140 Gly Asn Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala 145 150 155 160 Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Gln Asn Gln Gly Glu 165 170 175 Val Val Lys His Tyr Lys Ile Arg Asn Leu Asp Asn Gly Gly Phe Tyr 180 185 190 Ile Ser Pro Arg Ile Thr Phe Pro Gly Leu His Glu Leu Val Arg His 195 200 205 Tyr Thr Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys 210 215 220 Gln Thr Gln Lys Pro Gln Lys Pro Trp Trp Glu Asp Glu Trp Glu Val 225 230 235 240 Pro Arg Glu Thr Leu Lys Leu Val Glu Arg Leu Gly Ala Gly Gln Phe 245 250 255 Gly Glu Val Trp Met Gly Tyr Tyr Asn Gly His Thr Lys Val Ala Val 260 265 270 Lys Ser Leu Lys Gln Gly Ser Met Ser Pro Asp Ala Phe Leu Ala Glu 275 280 285 Ala Asn Leu Met Lys Gln Leu Gln His Gln Arg Leu Val Arg Leu Tyr 290 295 300 Ala Val Val Thr Gln Glu Pro Ile Tyr Ile Ile Thr Glu Tyr Met Glu 305 310 315 320 Asn Gly Ser Leu Val Asp Phe Leu Lys Thr Pro Ser Gly Ile Lys Leu 325 330 335 Thr Ile Asn Lys Leu Leu Asp Met Ala Ala Gln Ile Ala Glu Gly Met 340 345 350 Ala Phe Ile Glu Glu Arg Asn Tyr Ile His Arg Asp Leu Arg Ala Ala 355 360 365 Asn Ile Leu Val Ser Asp Thr Leu Ser Cys Lys Ile Ala Asp Phe Gly 370 375 380 Leu Ala Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg Glu Gly Ala 385 390 395 400 Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ile Asn Tyr Gly Thr 405 410 415 Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu 420 425 430 Ile Val Thr His Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu 435 440 445 Val Ile Gln Asn Leu Glu Arg Gly Tyr Arg Met Val Arg Pro Asp Asn 450 455 460 Cys Pro Glu Glu Leu Tyr Gln Leu Met Arg Leu Cys Trp Lys Glu Arg 465 470 475 480 Pro Glu Asp Arg Pro Thr Phe Asp Tyr Leu Arg Ser Val Leu Glu Asp 485 490 495 Phe Phe Thr Ala Thr Glu Gly Gln Tyr Gln Pro Gln Pro 500 505 499 amino acids amino acid unknown unknown None not provided 19 Met Gly Leu Leu Ser Ser Lys Arg Gln Val Ser Glu Lys Gly Lys Gly 1 5 10 15 Trp Ser Pro Val Lys Ile Arg Thr Gln Asp Lys Ala Pro Pro Pro Leu 20 25 30 Pro Pro Leu Val Val Phe Asn His Leu Ala Pro Pro Ser Pro Asn Gln 35 40 45 Asp Pro Asp Glu Glu Glu Arg Phe Val Val Ala Leu Phe Asp Tyr Ala 50 55 60 Ala Val Asn Asp Arg Asp Leu Gln Val Leu Lys Gly Glu Lys Leu Gln 65 70 75 80 Val Leu Arg Ser Thr Gly Asp Trp Trp Leu Ala Arg Ser Leu Val Thr 85 90 95 Gly Arg Glu Gly Tyr Val Pro Ser Asn Phe Val Ala Pro Val Glu Thr 100 105 110 Leu Glu Val Glu Lys Trp Phe Phe Arg Thr Ile Ser Arg Lys Asp Ala 115 120 125 Glu Arg Gln Leu Leu Ala Pro Met Asn Lys Ala Gly Ser Phe Leu Ile 130 135 140 Arg Glu Ser Glu Ser Asn Lys Gly Ala Phe Ser Leu Ser Val Lys Asp 145 150 155 160 Ile Thr Thr Gln Gly Glu Val Val Lys His Tyr Lys Ile Arg Ser Leu 165 170 175 Asp Asn Gly Gly Tyr Tyr Ile Ser Pro Arg Ile Thr Phe Pro Thr Leu 180 185 190 Gln Ala Leu Val Gln His Tyr Ser Lys Lys Gly Asp Gly Leu Cys Gln 195 200 205 Lys Leu Thr Leu Pro Cys Val Asn Leu Ala Pro Lys Asn Leu Trp Ala 210 215 220 Gln Asp Glu Trp Glu Ile Pro Arg Gln Ser Leu Lys Leu Val Arg Lys 225 230 235 240 Leu Gly Ser Gly Gln Phe Gly Glu Val Trp Met Gly Tyr Tyr Lys Asn 245 250 255 Asn Met Lys Val Ala Ile Lys Thr Leu Lys Glu Gly Thr Met Ser Pro 260 265 270 Glu Ala Phe Leu Gly Glu Ala Asn Val Met Lys Thr Leu Gln His Glu 275 280 285 Arg Leu Val Arg Leu Tyr Ala Val Val Thr Arg Glu Pro Ile Tyr Ile 290 295 300 Val Thr Glu Tyr Met Ala Arg Gly Cys Leu Leu Asp Phe Leu Lys Thr 305 310 315 320 Asp Glu Gly Ser Arg Leu Ser Leu Pro Arg Leu Ile Asp Met Ser Ala 325 330 335 Gln Val Ala Glu Gly Met Ala Tyr Ile Glu Arg Met Asn Ser Ile His 340 345 350 Arg Asp Leu Arg Ala Ala Asn Ile Leu Val Ser Glu Thr Leu Cys Cys 355 360 365 Lys Ile Ala Asp Phe Gly Leu Ala Arg Ile Ile Asp Ser Glu Tyr Thr 370 375 380 Ala Gln Glu Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala 385 390 395 400 Ile His Phe Gly Val Phe Thr Ile Lys Ala Asp Val Trp Ser Phe Gly 405 410 415 Val Leu Leu Met Val Ile Val Thr Tyr Gly Arg Val Pro Tyr Pro Gly 420 425 430 Met Ser Asn Pro Glu Val Ile Arg Ser Leu Glu His Gly Tyr Arg Met 435 440 445 Pro Cys Pro Glu Thr Cys Pro Pro Glu Leu Tyr Asn Asp Ile Ile Thr 450 455 460 Glu Cys Trp Arg Gly Arg Pro Glu Glu Arg Pro Thr Phe Glu Phe Leu 465 470 475 480 Gln Ser Val Leu Glu Asp Phe Tyr Thr Ala Thr Glu Gly Gln Tyr Glu 485 490 495 Leu Gln Pro 28 base pairs nucleic acid unknown unknown not provided 20 GGAATTCCCA CAGNGACTTN GCNGCNAG 28 26 base pairs nucleic acid unknown unknown cDNA not provided 21 GGAATTCCGA ANGTCCANAC GTCNGA 26 

What is claimed is:
 1. An isolated protein comprising the amino acid sequence shown in SEQ ID NO:4.
 2. An isolated protein comprising at least one of the following peptide domains contained within the amino acid sequence depicted in SEQ ID NO:4: a Pleckstrin homology peptide domain; an SH2 domain; an SH3 domain; and a catalytic domain.
 3. An isolated protein comprising at least one of the following amino acid sequences depicted in SEQ ID NO:4: amino acid residues 25 to 169; amino acid residues 296 to 375; amino acid residues 192-234; and amino acid residues 424 to
 659. 4. A fusion protein comprising the isolated protein of claim 1 or 2 fused to a heterologous amino acid sequence.
 5. An isolated protein comprising amino acid residues 660-675 of amino acid sequence SEQ ID NO:4.
 6. An isolated protein which is encoded by a naturally occurring nucleic acid molecule which hybridizes under highly stringent conditions to the nucleic acid sequence which encodes the polypeptide of SEQ ID NO:4, wherein said stringent conditions are selected from the group consisting of: (a) 0.15 M NaCl/0.0015 M sodiumcitrate/0.1% SDS at 50° C. for washing; (b) 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl and 75 mM sodium citrate at 42° C. during hybridization; or (c) hybridzation in 50% formamide, 5×SSC, 5×Denhardt's solution, 50 g/ml sonicated salmon sperm DNA, 0.1% SDS, and 10% dextran sulfate at 42°, with washes at 42° in 0.2×SSC and 0.1% SDS.
 7. An isolated protein comprising the amino acid sequence shown in SEQ ID NO:6.
 8. An isolated protein comprising at least one of the following peptide domains contained within the amino acid sequence depicted in SEQ ID NO:6: an SH2 domain; an SH3 domain; and a catalytic domain.
 9. An isolated protein comprising at least one of the following amino acid sequences depicted in SEQ ID NO:6: amino acid residues 122 to 201; amino acid residues 54 to 112; and amino acid residues 247 to
 486. 10. A fusion protein comprising the isolated protein of claim 7 or 8 fused to a heterologous amino acid sequence.
 11. An isolated protein which is encoded by a naturally occurring nucleic acid molecule which hybridizes under highly stringent conditions to the nucleic acid sequence which encodes the polypeptide of SEQ ID NO:6, wherein said stringent conditions are selected from the group consisting of: (a) 0.15 M NaCl/0.0015 M sodiumcitrate/0.1% SDS at 50° C. for washing; (b) 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl and 75 mM sodium citrate at 42° C. during hybridization; or (c) hybridzation in 50% formamide, 5×SSC, 5×Denhardt's solution, 50 g/ml sonicated salmon sperm DNA, 0.1% SDS, and 10% dextran sulfate at 42°, with washes at 42° in 0.2×SSC and 0.1% SDS. 